Methods and compositions for enhancing immunoassays

ABSTRACT

Embodiments of the methods, compositions, and systems provided herein relate to enzymatic enhancement of immunoassays using photonic sensor arrays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/964,899 filed Apr. 27, 2018 which is a division of U.S. applicationSer. No. 14/209,746 filed Mar. 13, 2014 now U.S. Pat. No. 9,983,206which issued May 29, 2018 which claims priority to U.S. Prov. App. No.61/788,279 filed on Mar. 15, 2013 entitled “ENZYMATIC ENHANCEMENT OFIMMUNOASSAYS FOR ULTRASENSITIVE DETECTION USING PHOTONIC SENSOR ARRAYS”the contents of which are each incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with United States Government support underGrant No. NSF CHE 12-14081 awarded by the National Science Foundation.The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

Embodiments of the methods, compositions, and systems provided hereinrelate to enzymatic enhancement of immunoassays using photonic sensorarrays.

BACKGROUND OF THE INVENTION

Robust biomolecule quantitation is central to biomarker based clinicaldiagnostics, driving the development of high throughput, low costmedical diagnostic devices based on a myriad of biosensing technologies.Chief among the many relevant performance metrics of these devices isthe ability to quantitate extremely low abundance analytes, such aspicograms per milliliter and less, in complex matrices and in amultiplexed format (Heath, J. R.; Davis, M. E. Annu. Rev. Med. 2008, 59,251-265). Regardless of the specific architecture or transductionmethodology, affinity-based biosensors face limitations imposed by theLangmuir binding isotherm, which defines the ratio of solution-phaseanalyte to surface-bound analyte, as determined by the affinity of thecapture agent employed. FIG. 1. At low concentrations, the amount ofbound analyte is directly proportional to the solution sampleconcentration, as shown in eq 1, where θ_(eq) is the equilibrium surfacecoverage, K_(ads) is the equilibrium binding constant, and [C] is thesolution-phase analyte concentration:

$\begin{matrix}{\theta_{eq} = \frac{K_{ads}\lbrack C\rbrack}{1 + {K_{ads}\lbrack C\rbrack}}} & \lbrack 1\rbrack\end{matrix}$

Even when using high-affinity capture agents, restrictions imposed bythe Langmuir isotherm, which are further exacerbated by mass transportlimitations for sensing elements with small geometric footprints, canresult in only a few individual molecules being bound to the sensorsurface (Squires, T. M.; Messinger, R. J.; Manalis, S. R. Nat.Biotechnol. 2008, 26, 417-426; Sheehan, P. E.; Whitman, L. J. Nano Lett.2005, 5, 803-807). Efforts to circumvent these fundamental limitations,and thus improve detection limits, include both the development ofnanostructured morphologies with increased surface areas, as well as theintegration of signal enhancement schemes that boost the per targetsensor response (Soleymani, L.; Fang, Z.; Lam, B.; Bin, X.; Vasilyeva,E.; Ross, A. J.; Sargent, E. H.; Kelley, S. O. ACS Nano 2011, 5,3360-3366; Munge, B. S.; Coffey, A. L.; Doucette, J. M.; Somba, B. K.;Malhotra, R.; Patel, V.; Gutkind, J. S.; Rusling, J. F. Angew. Chem.,Int. Ed. 2011, 50, 7915-7918). Nonetheless, there remains an unmet needto develop assays to detect and quantitate extremely low abundanceanalytes.

SUMMARY OF THE INVENTION

Some embodiments of the methods, compositions and systems providedherein include a method of detecting a target analyte comprising: (a)obtaining a planar substrate comprising an optical sensor having a firstcapture probe attached thereto; (b) contacting the first capture probewith a sample comprising a target analyte that selectively binds to thefirst capture probe; (c) contacting the bound target analyte with asecond capture probe that selectively binds to a complex comprising thebound target analyte, wherein the second capture probe comprises acatalyst; (d) contacting the catalyst with a reagent under conditionswhere the reagent forms a precipitate in the presence of the catalyst;and (e) measuring a change in an optical property at the optical sensor,thereby detecting the target analyte.

Some embodiments of the methods, compositions and systems providedherein include a method of detecting a target analyte comprising: (a)obtaining a planar substrate comprising an optical sensor having a firstcapture probe attached thereto; (b) contacting the first capture probewith a sample comprising a target analyte that selectively binds to thefirst capture probe; (c) contacting the bound target analyte with asecond capture probe that selectively binds to a complex comprising thebound target analyte, wherein the second capture probe comprises acatalyst; (d) contacting the catalyst with a reagent under conditionswhere the reagent forms a precipitate in the presence of the catalyst;and (e) measuring a change in resonance wavelengths at the opticalsensor, thereby detecting the target analyte.

In some embodiments, the second capture probe is formed by contacting anaffinity molecule bound to a first affinity tag with a second affinitytag bound to the catalyst, wherein the second affinity tag selectivelybinds to the first affinity tag.

In some embodiments, the second capture probe comprises an affinitymolecule bound to the catalyst.

In some embodiments, the second capture probe is attached to a particle.

In some embodiments, the catalyst is attached to a particle.

Some embodiments of the methods, compositions and systems providedherein include a method of detecting a target analyte comprising: (a)obtaining a planar substrate comprising an optical sensor having a firstcapture probe attached thereto; (b) contacting the first capture probewith a sample comprising a target analyte that selectively binds to thefirst capture probe; (c) contacting the bound target analyte with asecond capture probe that selectively binds to a complex comprising thebound target analyte, wherein the second capture probe comprises a firstaffinity tag; (d) contacting the bound second capture probe with asecond affinity tag that selectively binds to the bound first affinitytag, wherein the second affinity tag comprises a catalyst; (e)contacting the catalyst with a reagent under conditions where thereagent forms a precipitate in the presence of the catalyst; and (f)measuring an increase in precipitate formation at the optical sensor,thereby detecting the target analyte.

In some embodiments, the quantity of precipitate formation is indicativeof the level of the target analyte in the sample.

In some embodiments, an increase in rate of precipitate formation isindicative of the level of the target analyte in the sample.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, (f) comprises measuring a change in an opticalproperty.

In some embodiments, the change in an optical property is measured by aring resonator, and/or a wave guide structure.

In some embodiments, (f) comprises measuring a change in resonancewavelengths at the optical sensor.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe selectively binds to thetarget analyte.

In some embodiments, the second capture probe selectively binds to thebound target analyte.

In some embodiments, the second capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the secondcapture probe comprises an antibody selected from the group consistingof, anti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2from clone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe is attached to a particle.

In some embodiments, the catalyst is attached to a particle.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Insome embodiments, the catalyst comprises horseradish peroxidase.

In some embodiments, the reagent is selected from the group consistingof 4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole. In some embodiments, the reagent comprises4-chloro-1-naphthol.

Some embodiments also include contacting the catalyst with hydrogenperoxide. In some embodiments, the concentration of hydrogen peroxide isless than about 0.003%. In some embodiments, the concentration ofhydrogen peroxide is less than about 0.001%.

Some embodiments also include washing the optical sensor between steps(a)-(b), (b)-(c), (c)-(d), (d)-(e), and combinations thereof.

In some embodiments, a target analyte concentration less than about 100pg/ml is detected. In some embodiments, a target analyte concentrationless than about 10 pg/ml is detected. In some embodiments, a targetanalyte concentration less than about 1 pg/ml is detected.

In some embodiments, the target analyte comprises a cytokine. In someembodiments, the target analyte is selected from the group consisting ofIL-2, IL-4, IL-6, and IL-8.

In some embodiments, the sample comprises the target analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator.

In some embodiments, the optical sensor comprises a waveguide structure.

In some embodiments, the optical sensor comprises a well.

In some embodiments, the planar substrate comprises a plurality ofoptical sensors.

In some embodiments, the planar substrate comprises a thermal control.

In some embodiments, the planar substrate comprises an optical chip.

In some embodiments, the planar substrate comprises a multiwell plate.

In some embodiments, the planar substrate comprises a flowcell.

Some embodiments of the methods, compositions, and systems providedherein include a kit for detecting a target analyte comprising: a planarsubstrate comprising an optical sensor having a first capture probeattached thereto, wherein the first capture probe selectively binds tothe target analyte; a second capture probe that selectively binds to acomplex comprising the target analyte bound to the first capture probe,wherein the second capture probe comprises a catalyst; and a reagentthat can form a precipitate in the presence of the catalyst.

In some embodiments, the second capture probe is formed by contacting anaffinity molecule bound to a first affinity tag with a second affinitytag bound to the catalyst, wherein the second affinity tag selectivelybinds to the first affinity tag.

In some embodiments, the second capture probe comprises an affinitymolecule bound to the catalyst.

Some embodiments of the methods, compositions, and systems providedherein include a kit for detecting a target analyte comprising: a planarsubstrate comprising an optical sensor having a first capture probeattached thereto, wherein the first capture probe selectively binds tothe target analyte; a second capture probe that selectively binds to acomplex comprising the target analyte bound to the first capture probe,wherein the second capture probe comprises a first affinity tag; asecond affinity tag that selectively binds to the first affinity tag,wherein the second affinity tag comprises a catalyst; a reagent that canform a precipitate in the presence of the catalyst.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe selectively binds to thetarget analyte.

In some embodiments, the second capture probe selectively binds to thebound target analyte.

In some embodiments, the second capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the secondcapture probe comprises an antibody selected from the group consistingof, anti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2from clone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe is attached to a particle.

In some embodiments, the catalyst is attached to a particle.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

In some embodiments, the catalyst comprises horseradish peroxidase.

In some embodiments, the reagent is selected from the group consistingof 4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole. In some embodiments, the reagent comprises4-chloro-1-naphthol.

Some embodiments also include hydrogen peroxide. In some embodiments,the concentration of hydrogen peroxide is less than about 0.003%. Insome embodiments, the concentration of hydrogen peroxide is less thanabout 0.001%.

Some embodiments include a kit adapted to detect a target analyteconcentration less than about 100 pg/ml is detected. Some embodimentsinclude a kit adapted to detect a target analyte concentration less thanabout 10 pg/ml is detected. Some embodiments include a kit adapted todetect a target analyte concentration less than about 1 pg/ml isdetected.

In some embodiments, the target analyte comprises a cytokine. In someembodiments, the target analyte is selected from the group consisting ofIL-2, IL-4, IL-6, and IL-8.

In some embodiments, the sample comprises the target analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator.

In some embodiments, the optical sensor comprises a waveguide structure.

In some embodiments, the optical sensor comprises a well.

In some embodiments, the planar substrate comprises a plurality ofoptical sensors.

In some embodiments, the planar substrate comprises a thermal control.

In some embodiments, the planar substrate comprises an optical chip.

In some embodiments, the planar substrate comprises a multiwell plate.

In some embodiments, the planar substrate comprises a flowcell.

Some embodiments of the methods, compositions, and systems providedherein include a system for detecting a target analyte comprising: aplanar substrate comprising an optical sensor having a first captureprobe attached thereto, wherein the target analyte selectively binds tothe first capture probe; a second capture probe that selectively bindsto a complex comprising the target analyte bound to the first captureprobe, wherein the second capture probe comprises a catalyst; a reagentwhich can form a precipitate in the presence of the catalyst; and adetector adapted to measure a change in an optical property at theoptical sensor.

Some embodiments of the methods, compositions, and systems providedherein include a system for detecting a target analyte comprising: aplanar substrate comprising an optical sensor having a first captureprobe attached thereto, wherein the target analyte selectively binds tothe first capture probe; a second capture probe that selectively bindsto a complex comprising the target analyte bound to the first captureprobe, wherein the second capture probe comprises a catalyst; a reagentwhich can form a precipitate in the presence of the catalyst; and adetector adapted to measure a change in resonance wavelengths at theoptical sensor.

In some embodiments, a change in resonance wavelengths at the opticalsensor is indicative of the level of the target analyte.

In some embodiments, the second capture probe is formed by contacting anaffinity molecule bound to a first affinity tag with a second affinitytag bound to the catalyst, wherein the second affinity tag selectivelybinds to the first affinity tag.

In some embodiments, the second capture probe comprises an affinitymolecule bound to the catalyst.

Some embodiments of the methods, compositions, and systems providedherein include a system of detecting a target analyte comprising: aplanar substrate comprising an optical sensor having a first captureprobe attached thereto, wherein the target analyte selectively binds tothe first capture probe; a second capture probe that selectively bindsto a complex comprising the target analyte bound to the first captureprobe, wherein the second capture probe comprises a first affinity tag;a second affinity tag that selectively binds to the bound first affinitytag, wherein the second affinity tag comprises a catalyst; a reagentthat forms a precipitate in the presence of the catalyst; and a detectoradapted to measure an increase in precipitate formation.

In some embodiments, the increase in precipitate formation is indicativeof the level of the target analyte in the sample.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, an increase in precipitate formation is measured bya change in resonance wavelengths at the optical sensor.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe comprises an antibody orantigen-binding fragment thereof.

In some embodiments, the second capture probe selectively binds to thetarget analyte.

In some embodiments, the second capture probe selectively binds to thebound target analyte.

In some embodiments, the second capture probe comprises an antibodyselected from the group consisting of, anti-IL-2 from clone 555051,anti-IL-2 from clone 555040, anti-IL-2 from clone MQ1-17H12, anti-IL-6from clone BAF206, anti-IL-6 from clone MAB206, anti-IL-6 from cloneMQ2-13A5, anti-IL-6 from clone MQ2-39C3, and an antigen-binding fragmentthereof.

In some embodiments, the second capture probe is attached to a particle.

In some embodiments, the catalyst is attached to a particle.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Insome embodiments, the catalyst comprises horseradish peroxidase.

In some embodiments, the reagent is selected from the group consistingof 4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole. In some embodiments, the reagent comprises4-chloro-1-naphthol.

Some systems also include hydrogen peroxide.

In some embodiments, the planar surface is adapted for washing theoptical sensor.

In some embodiments, the system is adapted to detect a target analyteconcentration less than about 100 pg/ml is detected. In someembodiments, the system is adapted to detect a target analyteconcentration less than about 10 pg/ml is detected. In some embodiments,the system is adapted to detect a target analyte concentration less thanabout 1 pg/ml is detected.

In some embodiments, the target analyte comprises a cytokine. In someembodiments, the target analyte is selected from the group consisting ofIL-2, IL-4, IL-6, and IL-8.

In some embodiments, the sample comprises the target analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator.

In some embodiments, the optical sensor comprises a waveguide structure.

In some embodiments, the optical sensor comprises a well.

In some embodiments, the planar substrate comprises a plurality ofoptical sensors.

In some embodiments, the planar substrate comprises a thermal control.

In some embodiments, the planar substrate comprises an optical chip.

In some embodiments, the planar substrate comprises a multiwell plate.

In some embodiments, the planar substrate comprises a flowcell.

Some embodiments of the methods, compositions, and systems providedherein include a method of detecting a target analyte comprising: (a)obtaining an optical ring resonator having a first anti-cytokineantibody attached thereto, wherein the first anti-cytokine antibody isanti-IL-2 from clone 555051 antibody; (b) contacting the firstanti-cytokine antibody with a sample comprising IL-2 that selectivelybinds to the anti-cytokine antibody; (c) contacting the bound IL-2 witha second anti-cytokine antibody that selectively binds to the cytokine,wherein the second anti-cytokine antibody is a biotinylated anti-IL-2from clone 555040 antibody; (d) contacting the biolinylated secondanti-cytokine antibody with a streptavidin-horseradish peroxidaseconjugate; (e) contacting the horseradish peroxidase with4-chloro-1-naphthol and hydrogen peroxide under conditions that oxidize4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the4-chloro-1-naphthon precipitates on the surface of the optical ringresonator; and (f) measuring a change in resonance wavelengths of theoptical ring resonator, thereby indicating the presence of the cytokine.

In some embodiments, the concentration of hydrogen peroxide is less thanabout 0.003%.

In some embodiments, a concentration of the cytokine less than about 100pg/ml is detected.

Some embodiments of the methods, compositions, and systems providedherein include a method of detecting a target analyte comprising: (a)obtaining an optical ring resonator having a first anti-cytokineantibody attached thereto, wherein the first anti-cytokine antibody isanti-IL-6 from clone MAB206 antibody; (b) contacting the firstanti-cytokine antibody with a sample comprising IL-6 that selectivelybinds to the anti-cytokine antibody; (c) contacting the bound IL-2 witha second anti-cytokine antibody that selectively binds to the cytokine,wherein the second anti-cytokine antibody is a biotinylated anti-IL-6from clone BAF206; (d) contacting the biolinylated second anti-cytokineantibody with a streptavidin-horseradish peroxidase conjugate; (e)contacting the horseradish peroxidase with 4-chloro-1-naphthol andhydrogen peroxide under conditions that oxidize 4-chloro-1-naphthol to4-chloro-1-naphthon, whereby the 4-chloro-1-naphthon precipitates on thesurface of the optical ring resonator; and (f) measuring a change inresonance wavelengths of the optical ring resonator, thereby indicatingthe presence of the cytokine.

In some embodiments, the concentration of hydrogen peroxide is less thanabout 0.003%.

In some embodiments, a concentration of the cytokine less than about 100pg/ml is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph for the solution of calculations based on theLangmuir Binding Isotherm (Equation 1) which demonstrates the profoundeffect of capture agent affinity and analyte concentration on the numberof molecules bound to a sensor surface. It is preferable to use highgain amplification strategies to measure in the fM concentration rangedue to the minimal number of binding events. Assumptions include asensor surface area of 85 μm² and 6×10¹¹ capture agents/cm².

FIG. 2 shows a schematic diagram of an optical sensor comprising awaveguide and a ring resonator. FIG. 2 schematically illustrates therange of wavelengths that may be input into the optical sensor and theresultant spectral output of the optical sensor. A decrease in theoptical output at the resonance frequency of the ring resonator isvisible in the output spectrum shown

FIG. 3 is a cut-away view of a waveguide schematically showing anintensity distribution having an evanescent tail extending outside thewaveguide where an element such as a molecule or particle may be locatedso as to affect the index of refraction of the waveguide.

FIG. 4 depicts the horseradish peroxidase (HRP)-catalyzed oxidation of4-Cl-1-naphthol (4-CN) by hydrogen peroxide.

FIG. 5 depicts an HRP-amplified cytokine immunoassay on a microringresonator platform.

FIG. 6 depicts capture antibody loading with four-channel microfluidics.

FIG. 7 depicts screening of IL-2 antibody sandwich pairs.

FIG. 8 depicts a 100 pg/mL IL-2 sandwich assay with enzymaticamplification.

FIG. 9 depicts quenching of enzymatic deposition of 4-Cl-1-naphthonprecipitate by high hydrogen peroxide concentration.

FIG. 10 depicts large signal amplification with enzymatic amplificationgives nm-scale shifts down to 100 pg/mL IL-2.

FIG. 11 depicts calibration with four-step HRP enzymatic amplificationassay.

FIG. 12 depicts enzymatic amplification of IL-6 sandwich assay.

FIG. 13 depicts a typical sensor response for antibody immobilization tothe microring surface which resulted in a wavelength shift of between150-300 pm. It was determined that above 100 pm response, the amount ofantibody loading was independent of subsequent antigen, secondary, andtertiary responses.

FIG. 14 depicts a comparison of IL-6 quantitation by the method ofstandard additions for both ELISA and microring resonators

FIG. 15 depicts scanning electron micrographs of 3 different microringswith various IL-6 concentrations: a) 3125 pg/mL, b) 625 pg/mL, c)magnification of 625 pg/mL, d) 0 pg/mL. The heterogeneity of theprecipitation process is readily observed, likely corresponding tolocalized deposition around surface-immobilized HRP enzymes. The imageswere captured on a JEOL 6060LV general purpose SEM at 15 kV.

FIG. 16 depicts representative data corresponding to the inset abovewhich illustrates the large signal gain obtained via 4-chloro-1-naphthondeposition using an IL-6 concentration of 3125 pg/mL.

FIG. 17 depicts a scanning electron micrograph of an individualmicroring showing the discrete deposits of 4-CNP.

FIGS. 18A-18C depict the concentration dependent, real-time sensorresponse of 4-CN deposition observed to span a >3 order of magnitudedynamic range for all three interleukins studied. FIGS. 18D-18F depictthe associated calibration plots that illustrate a detection limit of 1pg/mL for all three interleukins and 500 fg/mL for IL-8. Calibrationplots are fit with logistic functions. Error bars represent the standarddeviation of n=3-4 individual sensors for microring measurements.

FIG. 19A depicts good agreement observed between ELISA and microringresonator based measurements of interleukin levels in CSF samples withthe use of an external standard method. Additionally, the two assaysshowed comparable precision. Error bars represent the standard deviationof n=3-4 individual sensors for microring measurements and n=3 forELISA. FIG. 19B depicts a focused study on IL-6 levels that showed evenbetter agreement with ELISA measurements when using a standard additioncalibration method; however, as expected, the precision for both ELISAand microring measurements is reduced when using the standard additionmethod.

DETAILED DESCRIPTION

Embodiments of the methods, compositions, and systems provided hereinrelate to enzymatic enhancement of immunoassays using photonic sensorarrays. Some such embodiments provide highly sensitive assays to detectand/or measure target analytes in a sample. In some embodiments, a firstcapture probe is attached to an optical ring resonator; a target analyteselectively binds to the first capture probe; a second capture probeselectively binds the complex comprising the bound target analyte. Insome embodiments, the second capture probe comprises a catalyst, such asan enzyme, such as horseradish peroxidase. In some embodiments, acatalyst selectively binds to the second capture probe through affinitytags. A reagent contacts the catalyst to form a precipitate. Theprecipitate is detected and measured by changes at the optical sensor.The use of two capture probes and a catalyst in the methods,compositions and systems provided herein greatly enhances any signal ofa target analyte binding to the first capture probe.

Beyond bound tag-based amplification strategies, enzymatic precipitatedeposition on the microrings represents another method for effectivelyamplifying the signal associated with analyte binding. Enzymaticreactions are commonly used in immunoassays, involving enzymes such ashorseradish peroxidase (HRP),^(1, 2) alkaline phosphatase,³ orβ-galactosidase⁴ to catalyze a chemical reaction that produces anoptical (usually colorimetric or fluorescent) readout. Theseenzyme-linked immunosorbent assays (ELISAs), which have been in use forover 30 years,⁵ have been useful for protein detection. Many ELISAs havebeen used to validate protein assays developed herein, with a multitudeof commercial products consistently demonstrating limits of detection inthe pg/mL range.^(6, 7) Enzymatic enhancement strategies are also highlyamenable to nucleic acid analyses and the detection of any species towhich the recognition of an enzymatically active moiety can befacilitated.

Though the ELISA offers excellent sensitivity and highly usefulmicrowell plate compatibility, the assay has many drawbacks. TraditionalELISAs are labor-intensive, with multiple washing steps and seeminglyendless pipetting, and this process is time-consuming (2-4 h).Additionally, traditional ELISAs are not a multiplexed technique: foreach analyte, a different microplate must be used. Though progress hasbeen made toward creating “mix-and-match” microwell strips that can beassembled into an array of microwells specific for different proteintargets, each analyte requires cumbersome and space-consumingindependent calibration wells. Even in the multiplexed ELISA format, asingle sample must be divided into multiple ELISA microwells and testedseparately for each analyte. Thus, ELISAs are fundamentally limited inmultiplexing capabilities by the sample volume (˜100 uL per well), thenumber of wells that a researcher is willing to employ for a singlestudy, and the logistics of conducting multiple independent ELISAs andseparate calibrations in parallel. These limitations can be overcome,while still maintaining pg/mL sensitivity, by combining microringresonators with enzymatic amplification.

While the use of enzymatic amplification on microring resonatorsintroduces complexity it can substantially improve the limit ofdetection. Instead of using an enzymatic process to introduce acolorimetric or fluorescent signal, the assay can be used to produce aninsoluble precipitate that substantially alters the effective refractiveindex (RI) at the ring surface. With an enzyme-amplified sandwich assay,cytokine concentrations that were previously unable to be detected canbe observed with ease.

In this disclosure, progress toward the optimization of an HRP-catalyzedamplification scheme is presented. This enzymatic process is applied toanalytes including interleukin-2 (IL-2) and interleukin-6 (IL-6)detection. Two-step cytokine sandwich assays typically have limits ofdetection on the order of 10 pM⁹ (and previous bead-based assays on thesame platform were capable to detect 200 fM protein),¹⁰ the HRPamplification assay achieves detection of IL-2 down to 67 fM (1 pg/mL).Importantly, the enzymatic process can display a lower and morereproducible background than other bead-based assays, also removing theneed for time-consuming bead exchange directly before running theassay.¹⁰ The ability to quantitate cytokines in the 1-100 pg/mL range isvital for a range of challenging protein biosensing applications,including serum and cerebrospinal fluid (CSF) diagnostics. Additionally,previously lowest-achievable cytokine detection limits (100 pg/mL-1ng/mL) on the platform, which were barely observable (<1 pm shift) withother microring resonator sandwich assays, now produce large nm-scalewavelength shifts with enzymatic amplification.

For enhancement strategies, desirable characteristics include highsignal gain, cross-platform modularity, and a resistance to matrixeffects. Previous demonstrations of signal enhancement strategies haveincluded enzymatic, nucleic acid, electrochemical, and particle-basedlabels (Munge, B. S.; Coffey, A. L.; Doucette, J. M.; Somba, B. K.;Malhotra, R.; Patel, V.; Gutkind, J. S.; Rusling, J. F. Angew. Chem.,Int. Ed. 2011, 50, 7915-7918; Konry, T.; Hayman, R. B.; Walt, D. R.Anal. Chem. 2009, 81, 5777-5782; Ivanov, I.; Stojcic, J.; Stanimirovic,A.; Sargent, E.; Nam, R. K.; Kelley, S. O. Anal. Chem. 2013, 85,398-403; (8) Luchansky, M. S.; Washburn, A. L.; McClellan, M. S.;Bailey, R. C. Lab Chip 2011, 11, 2042-2044; Krishnan, S.; Mani, V.;Wasalathanthri, D.; Kumar, C. V.; Rusling, J. F. Angew. Chem., Int. Ed.2011, 50, 1175-1178). Enzymatic-based methods are particularlyattractive due to the high gain possible from multiple turnovers.Horseradish peroxidase (HRP) has found usage in bioanalytical methods onaccount of its high substrate turnover (400 s⁻¹) and extended stability,which offers extremely high theoretical signal amplification (10⁷)(Veitch, N. C. Phytochemistry 2004, 65, 249-259; Azevedo, A. M.;Prazeres, D. M. F.; Cabral, J. M. S.; Fonseca, L. P. J. Mol. Catal. B:Enzym. 2001, 15, 147-153; Gorris, H. H.; Walt, D. R. J. Am. Chem. Soc.2009, 131, 6277-6282). Furthermore, numerous HRP-conjugates of usefulbiomolecular reagents are available commercially.

In this disclosure, an approach is described using a silicon photonicmicroring resonator detection platform, in which an HRP signalenhancement step is used to robustly provide limits of detection at orbelow 1 pg/mL, using three cytokines as representative biomoleculartargets. The enzymatic enhancement strategy described herein offerssuperior limits of detection while also featuring the potential forbroad applicability across multiple analyte types on account of itsmodular nature.

To showcase the pairing of microring resonators with this signalenhancement approach, the levels of three interleukins were quantitatedin both buffered solutions as well as undiluted cerebrospinal fluid.Interleukins are signaling cytokines that play a key role in regulatingimmune response but have also been implicated in the development andprogression of numerous diseases including Alzheimer's, dementia, andcancer. They are challenging analytes due to their naturally lowabundance, and undiluted CSF represents a complex and clinicallyrelevant matrix for robust assay validation (Sokolova, A.; Hill, M. D.;Rahimi, F.; Warden, L. A.; Halliday, G; M.; Shepherd, C. E. BrainPathol. 2009, 19, 392-398; Llano, D. A.; Li, J. H.; Waring, J. F.;Ellis, T.; Devanarayan, V.; Witte, D. G.; Lenz, R. A. Alzheimer Dis.Assoc. Disord. 2012, 26, 322-328; Angelopoulos, P.; Agouridaki, H.;Vaiopoulos, H.; Siskou, E.; Doutsou, K.; Costa, V.; Baloyiannis, S. I.Int. J. Neurosci. 2008, 118, 1659-1672; Allen, C.; Duffy, S.; Teknos,T.; Islam, M.; Chen, Z.; Albert, P. S.; Wolf, G.; Van Wales, C. Clin.Cancer Res. 2007, 13, 3182-3190; Anderson, N. L.; Anderson, N. G. Mol.Cell. Proteomics 2002, 1, 845-867). While enzyme-linked immunosorbentassays (ELISAs) are a method for interleukin quantitation, the laborintensive nature and limited multiplexing capacity of ELISA havemotivated the development of alternative approaches (Palandra, J.;Finelli, A.; Zhu, M.; Masferrer, J.; Neubert, H. Anal. Chem. 2013, 85,5522-5529; Luchansky, M. S.; Bailey, R. C. J. Am. Chem. Soc. 2011, 133,20500-20506). Combined with a horseradish peroxidase enzymatic signalenhancement strategy, we demonstrate that silicon photonic microringresonators can robustly quantitate biomarker concentrations in arelatively rapid and multiplexed assay format with limits of detectionat or below 1 pg/mL.

Sensor chip design and scanning instrumentation (Maverick detectionplatform from Genalyte, Inc.) and their use in the quantitation of arange of biomolecular targets, including proteins have been described(Washburn, A. L.; Gunn, L. C.; Bailey, R. C. Anal. Chem. 2009, 81,9499-9506; Iqbal, M.; Gleeson, M. A.; Spaugh, B.; Tybor, F.; Gunn, W.G.; Hochberg, M.; Baehr-Jones, T.; Bailey, R. C.; Gunn, L. C. IEEE J.Sel. Top. Quantum Electron. 2010, 16, 654-661). Some embodiments of themethods, compositions and systems provided herein include the use ofsilicon photonic resonators. In an example of a silicon photonicresonator, a sensor chip includes 32 microring sensor elements thatinclude 24 active microrings and 8 thermal controls. Microfluidicgaskets are used to spatially functionalize chips with up to fourdifferent analyte-specific capture antibodies. Target analytes arecaptured from the samples of interest as they are flowed across thesensor surface, and responses are enhanced with biomolecularspecifically through subsequent recognition with secondary antibodiesand tertiary reagents. All of these binding responses are measured byrecording the shifts in resonance wavelengths supported by the microringsensor elements, which are sensitive to the local refractive indexsampled by the circulating optical mode.

Silicon photonic resonators confine discrete frequencies of light viatotal internal reflection, and achieve constructive interference whenlight circumnavigates the structure an integer multiple of itswavelength, as shown in Equation 2, where m is a non-zero integer, r isthe microring radius and n_(eff) is the effective refractive indexsampled by the optical mode.

mλ=2πrn _(eff)   [2]

Boundary conditions of this propagating light dictate a non-zeroelectric field at the reflecting boundary, resulting in an evanescentfield extending into the local environment of the sensor. Interactionsbetween this evanescent field and the local environment modulate theresonant wavelength of the structure, which is monitored withsub-picometer precision by the optical scanning instrumentation.Biomolecule binding events result in the displacement of water(refractive index ˜1.33) with biomolecules of a higher refractive index(˜1.5), resulting in a positive shift in the resonant wavelength. Atunable external cavity diode laser centered at 1550 nm is coupled intothe sensor via microring-specific grating couplers, and scanned acrossan appropriate spectral window to determine transmission dips associatedwith resonant coupling. Sensor array elements are sequentially probed byrastering the laser across the substrate with each individual elementbeing interrogated approximately every 8 seconds, which is sufficient todirectly observe the kinetics of biomolecular binding.

As refractive index responsive devices, microring resonators canfunction as labelfree biochemical sensors. During the assay developmentprocess described herein, it was determined that sensor response wasfurther enhanced when using two secondary detection antibodies. In someembodiments, two detection antibodies were used for a target analyte.The secondary capture agent also provides an opportunity fortertiary-enhancement reagents to be included to achieve even largerper-analyte responses.

Optical Sensors

Some embodiments of the methods, compositions and systems providedherein include an optical sensor. Examples of optical sensors areprovided in U.S. Pub. No. 2013/0295688 which is incorporated herein byreference in its entirety. In some embodiments, optical sensors includesilicon photonic microring resonators which can have high spectralsensitivity towards surface binding events between a target analyte andan optical sensor modified with a capture probe that can selectivelybind to the target analyte. The systems of several embodiments are basedon refractive index-based sensing schemes in which the mass of boundanalytes, potentially in combination with other factors such as captureprobe affinity and surface density, contributes to the observed signaland measurement sensitivity.

In some embodiments, analyte detection can be accomplished using anoptically based system that includes a light source, an optical sensor,and an optical detector. In various embodiments, the light sourceoutputs a range of wavelengths. For example, the light source may be arelatively narrow-band light source that outputs light having a narrowbandwidth wherein the wavelength of the light source is swept over aregion many times the bandwidth of the light source. This light sourcemay, for example, be a laser. This laser may be a tunable laser suchthat the wavelength of the laser output is varied. In some embodimentsthe laser is a diode laser having an external cavity. This laser neednot be limited to any particular kind and may, for example, be a fiberlaser, a solid state laser, a semiconductor laser or other type of laseror laser system. The laser itself may have a wavelength that isadjustable and that can be scanned or swept. Alternatively, additionaloptical components can be used to provide different wavelengths. In someembodiments, the light source outputs light having a wavelength forwhich the waveguide structure is sufficiently optically transmissive. Insome embodiments, the waveguide structure is within a sample medium suchas an aqueous medium and the light source outputs light having awavelength for which the medium is substantially optically transmissivesuch that resonance can be reached in the optical resonator.Additionally, in some embodiments, the light source output has awavelength in a range where the analyte (e.g., molecules) of interest donot have a non-linear refractive index. Likewise, in variousembodiments, the light source may be a coherent light source thatoutputs light having a relatively long coherence length. However, invarious embodiments, the light source may be a coherent light sourcethat outputs light having a short coherence length. For example, incertain embodiments, a broadband light source such as asuper-luminescent light emitting diode (SLED) may be used. In suchcases, the wavelength need not be swept. An erbium amplifier runningbroadband that produces light having a range of wavelengths all at oncemay also be used. Light from the broadband source extending over anextended spectral range may be injected into the waveguide input. Aspectral analyzer (e.g., comprising a spectrometer) may be employed tocollect light from the waveguide output and analyze the output spectrum.

The light source provides light to the optical sensor. The light sourcemay be controlled by control electronics. These electronics may, forexample, control the wavelength of the light source, and in particular,cause the light source to sweep the wavelength of the optical outputthereof. In some embodiments, a portion of the light emitted from thelight source is sampled to determine, for example, the emissionwavelength of the light source.

In some embodiments, the optical sensor comprises a transducer thatalters the optical output based on the presence and/or concentration ofthe analyte to be detected. The optical sensor may be a waveguidestructure. The optical sensor may be an integrated optical device andmay be included on a chip. The optical sensor may comprise semiconductormaterial such as silicon. The optical sensor may be an interferometricstructure (e.g., an interferometer) and produce an output signal as aresult of optical interference. The optical sensor 104 may be includedin an array of optical sensors.

The optical detector detects the optical output of the sensor. Invarious embodiments, the optical detector comprises a transducer thatconverts an optical input into an electrical output. This electricaloutput may be processed by processing electronics to analyze the outputof the sensor. The optical detector may comprise a photodiode detector.Other types of detectors may be employed. Collection optics in anoptical path between the sensor and the detector may facilitatecollection of the optical output of the sensor and direct this output tothe detector. Additional optics such as mirrors, beam-splitters, orother components may also be included in the optical path from thesensor to the detector.

In various embodiments, the optical sensor is disposed on a chip whilethe light source and/or the optical detector are separate from the chip.The light source and optical detector may, for example, be part of anapparatus comprising free space optics that interrogates the opticalsensors on the chip.

In various embodiments, a solution such as an analyte solution is flowedpast the optical sensor. The detector detects modulation in an opticalsignal from the optical sensor when an analyte of interest is detected.

Ring resonators offer highly sensitive optical sensors that can beprepared so as to detect analytes. The operation of a ring resonator isshown in connection with FIG. 2. In this configuration, the opticalsensor comprises an input/output waveguide 202 having an input 204 andan output 206 and a ring resonator 208 disposed in proximity to aportion of the input/output waveguide 202 that is arranged between theinput 204 and the output 206. The close proximity facilitates opticalcoupling between the input/output waveguide 202 and the ring resonator208, which is also a waveguide. In this example, the input/outputwaveguide 202 is linear and the ring resonator 208 is circular such thatlight propagating in the input/output waveguide 202 from the input 204to the output 206 is coupled into the ring resonator 208 and circulatestherein. Other shapes for the input/output waveguide 202 (for example,curved) and ring resonator 208 (e.g., oval, elliptical, triangular,etc.) are also possible.

FIG. 2 shows an input spectrum 210 to represent that the light injectedinto the waveguide input 204 includes a range of wavelengths, forexample, from a narrow band light source having a narrow band peak thatis swept over time (or from a broadband light source such as asuper-luminescent diode). Similarly, an output spectrum 212 is shown atthe waveguide output 206. A portion of this output spectrum 212 isexpanded into a plot of intensity versus wavelength 214 and shows a dipor notch in the spectral distribution at the resonance wavelength, λ₀,of the ring resonator 208.

Without subscribing to any particular scientific theory, light“resonates” in the ring resonator when the number of wavelengths aroundthe ring (e.g. circumference) is exactly an integer. In this example,for instance, at particular wavelengths, light circulating in the ringresonator 208 is at an optical resonance when:

mλ=2πrn

-   -   where m is an integer, λ is the wavelength of light, r is the        ring radius, and n is the refractive index.

In this resonance condition, light circulating in the ring interfereswith light propagating within the linear waveguide 202 such that opticalintensity at the waveguide output 206 is reduced. Accordingly, thisresonance will be measured as an attenuation in the light intensitytransmitted down the linear waveguide 202 past the ring resonator 208 asthe wavelength is swept by the light source in a manner such as shown inthe plot 214 of FIG. 2.

Notably, the plot 214 in FIG. 2 shows the dip or notch having a width,δυ as measured at full width half maximum (FWHM) and an associatedcavity Q or quality factor, Q=λ₀/δυ. The ring resonator 208 produces arelatively high cavity Q and associated extinction ratio (ER) thatcauses the optical sensor 104 to have a heightened sensitivity.

As is well known, light propagates within waveguides via total internalreflection. The waveguide supports modes that yield a spatially varyingintensity pattern across the waveguide. A cross-section of a waveguide602 shown in FIG. 3 illustrates an example intensity distribution 604. Aplot 606 of the intensity distribution at different heights is providedadjacent the waveguide structure 602. As illustrated, a portion 608 ofthe electric field and optical energy referred to as the evanescent“tail” lies outside the bounds of the waveguide 602. The length of thisfield 608, as measured from the 1/e point, is between 50 and 150 nm,e.g. about 100 nm in some cases. An object 610 located close to thewaveguide 602, for example, within this evanescent field length affectsthe waveguide. In particular, objects 610 within this close proximity tothe waveguide 602 affect the index of refraction of the waveguide. Theindex of refraction, n, can thus be different when such an object 610 isclosely adhered to the waveguide 602 or not. In various embodiments, forexample, the presence of an object 610 increases the refractive index ofthe waveguide 602. In this manner, the optical sensor may be perturbedby the presence of an object 610 in the vicinity of the waveguidestructure 602 thereby enabling detection. In various embodiments, thesize of the object is about the length (e.g. 1/e distance) of theevanescent field to enhance interaction therebetween.

In the case of the ring resonator, an increase in the refractive index,n, increases the optical path length traveled by light circulating aboutthe ring. Longer wavelengths can resonate in the resonator and, hence,the resonance frequency is shifted to a lower frequency. The shift inthe resonant wavelengths of the resonator can therefore be monitored todetermine if an object 610 has located itself within close proximity tothe optical sensor (e.g., the ring resonator and/or a region of thelinear waveguide closest to the ring resonator). A binding event,wherein an object 610 binds to the surface of the optical sensor canthus be detected by obtaining the spectral output from the waveguideoutput and identifying dips in intensity (or peaks in attenuation)therein and the shift of these dips in intensity.

In various embodiments, the waveguide 602, e.g., the linear waveguideand/or the ring resonator comprise silicon. In some embodiments, thesurface of the waveguide 602 may be natively passivated with silicondioxide. As a result, standard siloxane chemistry may be an effectivemethod for introducing various reactive moieties to the waveguide 602,which are then subsequently used to covalently immobilize biomoleculesvia a range of standard bioconjugate reactions.

Moreover, the linear waveguide, ring resonator, and/or additionalon-chip optics may be easily fabricated on relatively cheapsilicon-on-insulator (SOI) wafers using well established semiconductorfabrication methods, which are extremely scalable, cost effective, andhighly reproducible. Additionally, these devices may be easilyfabricated and complications due to vibration are reduced when comparedto “freestanding” cavities. In one example embodiment, 8″ SOI wafers mayeach contain about 40,000 individually addressable ring resonators. Oneadvantage of using silicon-based technology is that various embodimentsmay operate in the Si transparency window of around 1.55 μm, a commonoptical telecommunications wavelength, meaning that lasers and detectorsare readily available in the commercial marketplace as plug-and-playcomponents.

Some embodiments of the waveguides useful with the methods, systems andcompositions provided herein include strip and rib waveguides. Othertypes of waveguides, such as for example, strip-loaded waveguides canalso be used. Lower cladding lies beneath the waveguides. In someembodiments, the waveguides are formed from a silicon-on-insulator chip,wherein the silicon is patterned to form the waveguides and theinsulator beneath provides the lower cladding. In many of theseembodiments, the silicon-on-insulator chip further includes a siliconsubstrate. Details on the fabrication of silicon biosensor chips can befound in Washburn, A. L., L. C. Gunn, and R. C. Bailey, AnalyticalChemistry, 2009, 81(22): p. 9499-9506, and in Bailey, R. C., Washburn,A. L., Qavi, A. J., Iqbal, M., Gleeson, M., Tybor, F., Gunn, L. C.Proceedings of SPIE—The International Society for Optical Engineering,2009, the disclosures of which are hereby incorporated by reference intheir entirety.

Still other designs than those specifically shown in the drawings hereinmay be employed. More ring resonators may be added. The resonators mayalso have different sizes and/or shapes. Additionally, the ringresonator(s) may be positioned differently with respect to each other aswell as with respect to the input/output waveguide. Likewise, morenon-ring resonator waveguides may be added.

In various embodiments, for example, a drop configuration is used. Forexample, in some such embodiments, a ring resonator is disposed betweenfirst and second waveguides. Light (such as a wavelength component) maybe directed into an input of the first waveguide and depending on thestate of the ring resonator, may be directed to either an output of thefirst waveguide or an output of the second waveguide. For example, forresonant wavelengths, the light may be output from the second waveguideinstead of the first waveguide. An optical detector may thus monitorshifts in intensity peaks to determine the presence of an analyte ofinterest detected by the optical sensor in some such embodiments.

Combinations of these different features are also possible. Moreover,multiple resonators and/or waveguides may be placed in any desiredgeometric arrangement. Additionally, spacing between resonators and/orwaveguides may be varied as desired. Different features can be combinedin different ways.

Also, although linear waveguides are shown in FIGS. 2 and 3 as providingaccess to the ring resonators, these waveguides need not be restrictedto plain linear geometry. In some examples, for instance, thesewaveguides may be curved or otherwise shaped differently. Likewise thering resonators need not be circularly shaped but can have other shapes.The ring resonators may be oval or elliptically-shaped,triangularly-shaped or irregularly shaped.

Other geometries may possibly be used for the resonator, such as, forexample, microsphere, microdisk, and microtoroid structures. See, e.g.,Vahala, Nature 2003, 424, 839-846; and in Vollmer & Arnold, NatureMethods 2008, 5, 591-596, the disclosures of which are herebyincorporated by reference in their entirety. Again, combinations ofthese different features are also possible and different features can becombined in different ways.

Additional details regarding sensors and apparatus for interrogatingsuch sensors are included in U.S. Patent Publication 2011/0045472 titled“Monitoring Enzymatic Process” as well as PCT Publication WO 2010/062627titled “Biosensors Based on Optical Probing and Sensing”, which are eachincorporated herein by reference in its entirety.

Target Analytes

Some embodiments of the methods, compositions and systems providedherein include a target analyte. As used herein a ‘target analyte’ caninclude a substance to be detected in a test sample. In someembodiments, a target analyte can include a nucleic acid, protein, and acarbohydrate. As used herein, a ‘protein’ can include a polypeptide, apeptide, hormones, enzyme, antibodies and fragments thereof. In someembodiments, a target analyte is an antigen that selectively binds to anantibody or antigen-binding fragment thereof. In some embodiments, atarget analyte includes a cytokine. Examples of cytokines includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. In some embodiments, cytokines include IL-2, IL-6, and IL-8.

In some embodiments, the target analyte is associated with a disordersuch as brain trauma, stroke, multiple sclerosis, post-traumatic stressdisorder, assorted infections of the central nervous system, andAlzheimer's disease.

Test Samples

Some embodiments of the methods, compositions and systems providedherein include a test sample. In some embodiments, a test sampleincludes a target analyte. In some embodiments, a test sample comprisesa biological sample. Examples of biological samples include anybiological tissue or fluid derived from a subject such as sputum,cerebrospinal fluid, blood, blood fractions such as serum and plasma,blood cells, tissue, biopsy samples, urine, peritoneal fluid, pleuralfluid, amniotic fluid, vaginal swab, skin, lymph fluid, synovial fluid,feces, tears, organs, or tumors. In some embodiments, a biologicalsample can include viral particles or fragments thereof, recombinantcells, cell components, cells grown in vitro, and cell cultureconstituents including, for example, conditioned medium resulting fromthe growth of cells in cell culture medium.

Capture Probes

Some embodiments of the methods, compositions and systems providedherein include a capture probe. A capture probe selectively binds to atarget analyte. In some embodiments, a capture probe can selectivelybind to a complex comprising a target analyte and another capture probe.In some embodiments, a capture probe can selectively bind to a targetanalyte bound to another capture probe. In some embodiments, a captureprobe is attached to an optical sensor. Examples of capture probesinclude antibodies and antigen-binding fragments thereof; polypeptides;nucleic acids; and lectins.

As used herein “antibody” can include synthetic antibodies, monoclonalantibodies, recombinantly produced antibodies, intrabodies,multispecific antibodies (including bi-specific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, syntheticantibodies, single-chain Fvs (scFv), Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv) (including bi-specific sdFvs), andanti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. In some embodiments, an antibody or antigen-bindingfragment thereof can be monospecific, bispecific, trispecific or ofgreater multispecificity. Multispecific antibodies may be specific fordifferent epitopes of a polypeptide or may be specific for both apolypeptide as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, etal., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992); each of which is incorporated herein by referencein its entirety.

In some embodiments, a capture probe can include a polypeptide, which isinclusive of known polypeptide analogs. Examples of polypeptide analogsinclude molecules that comprise a non-naturally occurring amino acid,side chain modification, backbone modification, N-terminal modification,and/or C-terminal modification known in the art. For example, apolypeptide capture probe can comprise a D-amino acid, a non-naturallyoccurring L-amino acid, such as L-(1-naphthyl)-alanine,L-(2-naphthyl)-alanine, L-cyclohexylalanine, and/or L-2-aminoisobutyricacid. In some embodiments, a polypeptide capture probe can comprise anantigen to which an antibody target analyte is capable of binding. Invarious aspects, a capture probe can comprise a polypeptide antigencapable of binding to an antibody of interest that is a known biomarkerfor a particular disease or condition. It will be appreciated that acapture probe of the systems provided herein can comprise any antigenassociated with any disease or condition for which a subject's antibodyagainst the antigen is considered a biomarker. As a non-limitingexample, a capture probe can comprise a viral antigen capable of bindingto an antibody specific against the viral antigen. Presence of such anantibody, as detected by the systems provided herein, would indicatethat the subject has been infected by the virus and mounted a specificimmune response to it. In certain embodiments, a capture probe cancomprise an auto-antigen associated with an autoimmune disorder or anantigen associated with an allergy, which capture probe is capable ofbinding to an antibody, such as an auto-antibody, of interest. Presenceof such an antibody, as detected by the systems provided herein, wouldindicate that the subject has or is at risk of having the associatedautoimmune disorder or allergy.

In some embodiments, a capture probe can include a nucleic acid. As usedherein with respect to capture probes, “nucleic acid” refers todeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and known analogs,derivatives, or mimetics thereof. A nucleic acid capture probe can beoligomeric and include oligonucleotides, oligonucleosides,oligonucleotide analogs, oligonucleotide mimetics and chimericcombinations of these. A nucleic acid capture probe can besingle-stranded, double-stranded, circular, branched, or hairpin and cancontain structural elements such as internal or terminal bulges orloops. In some embodiments, a nucleic acid capture probe can have alength of at least, or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleobases,or the nucleic acid capture probe can have a length within any rangebounded by two of the above-mentioned lengths.

In several embodiments, a nucleic acid capture probe and a targetanalyte comprising a nucleic acid bind to form a duplex. Such bindingmay occur through hybridization. As used herein, “hybridization” meansthe pairing of complementary strands of a nucleic acid capture probe anda nucleic acid analyte of interest. In some embodiments, a nucleic acidcapture probe and nucleic acid molecule of interest can hybridize under“stringent conditions,” which refer to conditions under which a nucleicacid capture probe will hybridize to a nucleic acid molecule ofinterest, but to a minimal number of other sequences. A person ofordinary skill in the art will appreciate that stringent conditions aresequence-dependent and will vary in different circumstances. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C.“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases of a nucleic acid capture probe andnucleic acid analyte of interest. For example, if a nucleobase at acertain position of a capture probe is capable of hydrogen bonding witha nucleobase at a certain position of a nucleic acid analyte ofinterest, then the position of hydrogen bonding between the captureprobe and the nucleic acid analyte of interest is considered to be acomplementary position. The capture probe and the analyte of interestare complementary to each other when a sufficient number ofcomplementary positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, in some embodiments anucleic acid capture probe and nucleic acid analyte of interest arespecifically hybridizable and complementary, which indicate a sufficientdegree of precise pairing or complementarity over a sufficient number ofnucleobases such that stable and specific binding occurs. It will beappreciated that the sequence of a nucleic acid capture probe need notbe 100% complementary to that of a nucleic acid analyte of interest tobe specifically hybridizable. Moreover, a nucleic acid capture probe mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In several embodiments, a nucleic acid capture probe can comprise one ormore oligonucleotide mimetics. The term “mimetic” includes oligomericnucleic acids wherein the furanose ring or the furanose ring and theinternucleotide linkage are replaced with non-naturally occurringgroups. In certain embodiments, a nucleic acid capture probe comprises apeptide nucleic acid (PNA) oligonucleotide mimetic (Nielsen et al.,Science, 1991, 254, 1497-1500). PNAs have favorable hybridizationproperties, high biological stability and are electrostatically neutralmolecules. Representative United States Patents that teach thepreparation of PNA oligomeric compounds include U.S. Pat. Nos.5,539,082; 5,714,331 and 5,719,262. PNA compounds can be obtainedcommercially from Applied Biosystems (Foster City, Calif., USA). In someembodiments, capture probes comprising nucleic acids can include anoligonucleotide mimetic such as a linked morpholino units (morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring(Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14),4503-4510). In some embodiments, capture probes comprising nucleic acidscan include an oligonucleotide mimetic such as a cyclohexene nucleicacids (CeNA) (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Insome embodiments, capture probes comprising nucleic acids can include alocked nucleic acid (LNA). An LNA capture probe includes nucleoside ornucleotide analogues that include at least one LNA monomer (e.g., an LNAnucleoside or LNA nucleotide). LNA monomers are described in, forexample, WO 99/14226, U.S. Pat. Nos. 6,043,060, 6,268,490, WO 01/07455,WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604. Inseveral embodiments, a nucleic acid capture probe can include anon-native, degenerate, or universal base such as inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thioLiracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine, or the like. In some embodiments, a nucleic acid captureprobe can include isocytosine and/or isoguanine in order to reducenon-specific hybridization as generally described in U.S. Pat. No.5,681,702.

In several embodiments, a nucleic acid capture probe can comprise an“aptamer” to bind to a nucleic acid or polypeptide analyte of interest.Aptamers are described in U.S. Pat. Nos. 5,270,163; 5,475,096;5,567,588; 5,595,877; 5,637,459; 5,683,867; and 5,705,337; which areherein incorporated by reference in their entireties. Aptamers can bindto various molecular targets such as small molecules, proteins, andnucleic acids.

In some embodiments, wherein the target analyte comprises acarbohydrate, suitable capture probes can include lectins. Lectins areproteins that bind to saccharides and differ in the types ofcarbohydrate structures they recognize. Several known lectins that canbe used in capture probes of various embodiments include those that havebeen isolated from plants including Conavalia ensiformis, Anguillaanguilla, Triticum vulgaris, Datura stramoniuim, Galanthus nivalis,Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythrinacristagalli, Lens culinaris, Glycine may, Phaseolus vulgaris, Allomyrinadichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, andRicinus communis. Additional lectins that can be used in capture probesof several embodiments include any of the animal, bacterial, or fungallectins known in the art. Several bacterial and fungal lectins haveconsiderably high affinity (micromolar Kd) towards carbohydratescompared to plant or animal lectins.

Attachment of Capture Probes to Optical Sensors

In some embodiments, a capture probe is attached to an optical sensor.Methods of attaching a capture probe to a substrate comprising anoptical sensor are described in U.S. Pub. No. 2013/0295688 which isincorporated herein by reference in its entirety. In some embodiments,the capture probes are attached to a surface of an optical sensor by alinkage, which may comprise any moiety, functionalization, ormodification of the binding surface and/or capture probes thatfacilitates the attachment of the capture probes to the surface of theoptical sensor. The linkage between the capture probes and the surfaceof the optical sensor can comprise one or more chemical bonds; one ormore non-covalent chemical bonds such as Van der Waals forces, hydrogenbonding, electrostatic interaction, hydrophobic interaction, orhydrophilic interaction; and/or chemical linkers that provide suchbonds.

Further Capture Probes

Some embodiments of the methods, compositions and systems providedherein include a second capture probe. In some embodiments, such captureprobes selectively bind to a complex comprising a target analyte boundto another capture probe. The second capture probe can comprise anycapture probe described herein. In some embodiments, the second captureprobe comprises an antibody or antigen-binding fragment thereof. In someembodiments, the second capture probe comprises a detectable label. Insome embodiments the detectable label comprises a catalyst, such as anenzyme.

Similar to a sandwich assay format in which an antigen is first bound bya substrate-immobilized primary capture agent and then recognized by asecondary capture agent, the systems of several embodiments providedherein comprise a capture probe (analogous to a sandwich assay primarycapture agent) and an antibody (analogous to a sandwich assay secondarycapture agent). It is possible to detect and/or measure binding-inducedshifts in the resonance wavelength of individual binding events with thesystems of various embodiments, including binding of an antibody to theoptical sensor. Without being bound by theory, binding of an antibody tothe optical sensor can induce a change in local refractive index,thereby inducing a detectable and/or measurable shift in the resonancewavelength on the optical sensor.

In several embodiments, a system for detecting and/or measuring ananalyte of interest includes an antibody capable of binding to theanalyte of interest or a complex or duplex formed between a captureprobe attached to a surface of an optical sensor and the analyte ofinterest. It will be understood that in several embodiments the antibodycapable of binding to a complex or duplex formed between a capture probeand analyte of interest can bind to a portion of the analyte of interestthat is not bound to the capture probe in formation of the complex orduplex such that the antibody does not directly bind and/or physicallycontact the capture probe. Thus, the binding of a capture probe/analytecomplex by the antibody can be accomplished by the antibody contactingand binding only the analyte portion of the capture probe/analytecomplex. In various aspects, an antibody can bind to an epitope on ananalyte of interest distinct from the epitope or binding site on theanalyte of interest involved in binding to the capture probe. In someaspects, the antibody capable of binding to a complex or duplex formedbetween a capture probe and analyte of interest binds to the analyte ofinterest without inhibiting or interfering with the binding between theanalyte of interest and the capture probe.

An example of a binding event that increases the refractive index at theoptical sensor surface and can be observed as an increase in theresonance wavelength of the optical sensor is an antibody-analytecomplex binding to a capture probe attached to a surface of an opticalsensor (a “primary” binding event). Yet another detectable and/ormeasurable binding event is an antibody binding to an analyte ofinterest which is already bound to a capture probe attached to a surfaceof an optical sensor (a “secondary” binding event). A further detectableand/or measurable binding event is an antibody binding to a duplex orcomplex formed between an analyte of interest and a capture probeattached to a surface of an optical sensor (a “secondary” bindingevent).

It will be understood by a person of ordinary skill in the art that inseveral aspects, an antibody can bind to the analyte of interest eitherprior to or after binding between the analyte of interest and captureprobe. Thus, in some embodiments a binding-induced shift in theresonance wavelength can be detected and/or measured for (1) anantibody-analyte complex binding to a capture probe attached to asurface on an optical sensor, (2) an antibody binding to the analytealready bound to the capture probe attached to a surface on an opticalsensor, or (3) an antibody binding to the duplex or complex formedbetween the analyte and capture probe attached to a surface on anoptical sensor. It will also be apparent to a person of ordinary skillin the art that in some aspects, an antibody is not capable of bindingto the capture probe alone or analyte of interest alone, but is capableof binding to the complex or duplex formed between the capture probe andanalyte of interest.

Accordingly, certain embodiments drawn to a system for detecting ananalyte of interest includes both (1) a capture probe comprising anantibody attached to a surface of an optical sensor and (2) an antibodycapable of binding to the analyte of interest either prior to or afterbinding between the analyte of interest and capture probe. In additionalembodiments, a system for detecting an analyte of interest includes (1)a capture probe comprising a nucleic acid attached to a surface of anoptical sensor wherein the capture probe is capable of binding to ananalyte of interest, and (2) an antibody that is not capable of bindingto the capture probe alone or analyte of interest alone, but is capableof binding to the complex or duplex formed between the capture probe andanalyte of interest.

In certain embodiments, the system includes an antibody thatspecifically binds to an oligonucleotide duplex, such as a DNA:RNAduplex, DNA:DNA duplex, or RNA:RNA duplex, formed between a captureprobe and analyte of interest, but does not bind to the nucleic acidcapture probe or analyte of interest prior to their binding. As usedherein, the term “duplex” refers to a double-stranded molecule, whichcan be formed by hybridization of single-stranded nucleic acids.

Anti-DNA:RNA antibodies can detect miRNA analytes of interest whilesignificantly reducing assay complexity. Both monoclonal and polyclonalantibodies against RNA:RNA and DNA:RNA homoduplexes have been previouslydeveloped and utilized in hybridization based assays for the detectionof numerous nucleic acid targets such as viral nucleic acids and E. colismall RNA. Casebolt, D. B. and C. B. Stephensen, Journal of ClinicalMicrobiology, 1992. 30(3): p. 608-12; Fliss, I., et al., Appl MicrobiolBiotechnol, 1995. 43(4): p. 717-24; Lafer, E. M., et al., J Biol Chem,1986. 261(14): p. 6438-43; Riley, R. L., D. J. Addis, and R. P. Taylor,J Immunol, 1980. 124(1): p. 1-7; Stollar, B. D., FASEB J, 1994. 8(3): p.337-42 and Stollar, B. D. and A. Rashtchian, Anal Biochem, 1987. 161(2):p. 387-94; which are all incorporated by reference in their entireties.

In particular embodiments, a system for detecting an analyte of interestincludes an antibody that specifically binds to a DNA:RNA duplex. Onenon-limiting example of such an antibody that can be used in severalembodiments is that specifically binds to a DNA:RNA duplex is S9.6, amonoclonal antibody that specifically binds to RNA-DNA hybrids asdescribed in Boguslawski et al., J. Immunological Methods, 89 (1986)123-130, which is herein incorporated by reference in its entirety.

Particles

Some embodiments of the methods, compositions and systems providedherein include particles. While systems comprising an antibodyconfigured in a sandwich assay format can detect and/or measure“primary” or “secondary” binding events, several embodiments are drawnto systems comprising a particle adapted to amplify a detectable and/ormeasurable optical property that is altered (e.g. resonance wavelength)upon a binding event on an optical sensor. Such embodiments are based onthe present discovery that a “secondary” or “tertiary” binding event ofparticles to an optical sensor can increase the sensitivity of detection(i.e. lower the detection limit) by several-fold. For example, aparticle can increase the sensitivity of detection from approximatelythe low pM to the high fM range, compared to a “secondary” bindingevent. In certain embodiments, systems can comprise a particle adaptedto provide a “primary” binding event detectable signal. For example, aparticle can be bound to an analyte of interest and a complex formedbetween them can then be bound to a capture probe attached to a surfaceof an optical sensor. In some embodiments, a particle can be bound to asecond capture probe. Examples of particles are described in U.S. Pub.No. 2013/0295688 which is incorporated herein by reference in itsentirety.

Several embodiments relate to a system for detecting an analyte ofinterest including a particle attached to an antibody, which is capableof specifically binding to the analyte or a duplex or complex formedbetween the analyte and capture probe, or capable of binding to theantibody. The particle is adapted to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor. In one aspect, a particle can bind to an antibody thatis already bound to an optical sensor, whether via binding to an analytewhich is bound to a capture probe attached to a surface of the opticalsensor or binding to a duplex or complex formed between the analyte ofinterest and a capture probe. Such a binding of the particle in thisfashion can be considered a “tertiary” binding event, while the priorbinding of the antibody to the optical sensor is a “secondary” bindingevent and the binding of the analyte of interest to the capture probe isa “primary” binding event.

In various embodiments, a particle can be associated with a molecule(e.g. by conjugation) that has affinity for the analyte of interest. Forexample, and not by limitation, a particle can be associated with asilane molecule having affinity to a polypeptide analyte of interest; aparticle can be associated with a phosphate-containing molecule havingaffinity to a nucleic acid analyte of interest; a particle can beassociated with a salt having affinity to a carbohydrate analyte ofinterest; or a particle can be associated with a organic molecule havingaffinity to a lipid.

It will be understood that in several aspects, a particle can beassociated with a molecule that has affinity for the analyte of interestin the same way that capture probes described above can bind to ananalyte of interest. For example, the analyte of interest and moleculeassociated with a particle can represent a binding pair, which caninclude but is not limited to antibody/antigen (nucleic acid orpolypeptide), receptor/ligand, polypeptide/nucleic acid, nucleicacid/nucleic acid, enzyme/substrate, carbohydrate/lectin, orpolypeptide/polypeptide. In some embodiments, a particle comprises acatalyst, such as an enzyme, such as horseradish peroxidase. It willalso be understood that binding pairs of analytes of interest andmolecules associated with particles described above can be reversed inseveral embodiments. Any of the functional groups and linkers describedabove with respect to attaching capture probes to an optical sensorsurface can be used to conjugate particles to molecules that haveaffinity to an analyte of interest. In certain embodiments, an antibodycan be conjugated to a particle, such as a COOH-functionalizedpolystyrene bead, via a n-hydroxysuccinimide ester (NHS) linkage, a DNAmolecule can be conjugated to a particle, such as a streptavidin coatedglass microsphere via biotin-streptavidin binding, a carbohydratemolecule can be conjugated to a particle, such as a gold nanoparticle,via a thiol linkage, a polypeptide molecule can be conjugated to aparticle, such as a titanium dioxide nanoparticle, via an isocyanatesilane linkage, and a polypeptide molecule can be conjugated to aparticle, such as a magnetic nanoparticle or microsphere, via1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). It will also beunderstood that in various embodiments a molecule that has affinity forthe analyte of interest can be associated with a particle by passiveabsorption.

It will be appreciated that a particle can comprise any material, shape,physical state, and/or size sufficient to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor. Without being bound by theory, in some embodiments aparticle comprises any material, shape, physical state, and/or sizesufficient to increase the refractive index at the sensor surface, whichcan be observed as an increase in the resonance wavelength of theoptical sensor. Any particle that has sufficient mass or other physicalproperty, such as electron density, to increase the refractive index atthe sensor surface can be used. In some embodiments, a particle can beamorphous or spherical, cubic, star-shaped, and the like. The particlesprovided herein can comprise solids, liquids, or gases. In severalembodiments, a particle can comprise crystalline, polycrystalline,polymer, glass, biopolymer, or a composite of these materials.

In some embodiments, a particle adapted to amplify a detectable and/ormeasurable optical property that is altered upon a binding event on anoptical sensor has a dimension along any axis, such as an averagediameter, of at least about 0.1 nanometers (nm), 0.5 nm, 1 nm, 5 nm, 10nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800nm, 900 nm, 1,000 nm, 2,000 nm, 3,000 nm, 4,000 nm, 5,000 nm, greaterthan 5,000 nm, any number in between the aforementioned dimensions, orany range between two of the aforementioned dimensions. In severalembodiments, a particle has a dimension along any axis, such as anaverage diameter, of about 1 nm to 1,000 nm. In several embodiments, aparticle has a dimension along any axis, such as an average diameter, ofabout 50 nm to 200 nm.

In some embodiments, a particle comprises a polypeptide of at least 200Daltons, (Da), 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1kilo Dalton (kDa), 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 50 kDa, 75kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800kDa, 900 kDa, 1,000 kDa, 2,000 kDa, 3,000 kDa, 4,000 kDa, 5,000 kDa,6,000 kDa, 7,000 kDa, 8,000 kDa, 9,000 kDa, 10,000 kDa, greater than10,000 kDa, or any size or range between any two of the aforementionedsizes.

In some embodiments, a particle comprises any known polypeptide commonlyused in molecular biology as recombinant expression or purification tagsincluding, but not limited to histidine (His), maltose binding protein(MBP), FLAG, Trx, myc, streptavidin, biotin, human influenza virushemagluttinin (HA), vesicular stomatitis virus glycoprotein (VSV-G),glycoprotein-D precursor of Herpes simplex virus (HSV), V5, AU1,glutathione-S-transferase (GST), the calmodulin binding domain of thecalmodulin binding protein, Protein A, and Protein G. Non-limitingexamples of specific protocols for selecting, making and using anappropriate tag are described in, e.g., Epitope Tagging, pp. 17.90-17.93(Sambrook and Russell, eds., Molecular Cloning A Laboratory Manual, Vol.3, 3rd ed. 2001), which is herein incorporated by reference in itsentirety.

In several embodiments, a particle comprises a nanoparticle, nanosphere,microcapsule, nanocapsule, microsphere, microparticle, bead, colloid,aggregate, flocculate, insoluble salt, emulsion, crystal, detergent,surfactant, dendrimer, copolymer, block polymer, nucleic acid,carbohydrate, lipid, liposome, or insoluble complex. It is contemplatedthat these types of particles can have any size in the picometer,nanometer, micrometer, or millimeter range along any dimensional axis.As used herein, the term “nanoparticle” refers to any particle having agreatest dimension (e.g., diameter) that is less than about 2500 nm. Insome embodiments, the nanoparticle is a solid or a semi-solid. In someembodiments, the nanoparticle is generally centrosymmetric. In someembodiments, the nanoparticle contains a generally uniform dispersion ofsolid components.

Nanoparticles can have a characteristic dimension of less than about 1micrometer, where the characteristic dimension of a particle is thediameter of a perfect sphere having the same volume as the particle. Forexample, the nanoparticle may have a characteristic dimension that isless than 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 180 nm, 150 nm, 120nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm,or any number in between the aforementioned sizes. In some embodiments,the nanoparticle can have a characteristic dimension of 10 nm, 20 nm, 30nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 150 nm,180 nm, 200 nm, 250 nm or 300 nm, or any number in between theaforementioned sizes. In other embodiments, the nanoparticle can have acharacteristic dimension of 10-500 nm, 10-400 nm, 10-300 nm, 10-250 nm,10-200 nm, 10-150 nm, 10-100 nm, 10-75 nm, 10-50 nm, 50-500 nm, 50-400nm, 50-300 nm, 50-200 nm, 50-150 nm, 50-100 nm, 50-75 nm, 100-500 nm,100-400 nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150 nm, 150-500 nm,150-400 nm, 150-300 nm, 150-250 nm, 150-200 nm, 200-500 nm, 200-400 nm,200-300 nm, 200-250 nm, 200-500 nm, 200-400 nm or 200-300 nm.

In various embodiments, a particle comprises one or more materialsincluding, but not limited to, polymers such as polystyrene, siliconerubber, latex, polycarbonate, polyurethanes, polypropylenes,polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene. Additional examples of suitable polymers include, but arenot limited to the following: polyethylene glycol (PEG); poly(lacticacid-co-glycolic acid) (PLGA); copolymers of PLGA and PEG; copolymers ofpoly(lactide-co-glycolide) and PEG; polyglycolic acid (PGA); copolymersof PGA and PEG; poly-L-lactic acid (PLLA); copolymers of PLLA and PEG;poly-D-lactic acid (PDLA); copolymers of PDLA and PEG; poly-D,L-lacticacid (PDLLA); copolymers of PDLLA and PEG; poly(ortho ester); copolymersof poly(ortho ester) and PEG; poly(caprolactone); copolymers ofpoly(caprolactone) and PEG; polylysine; copolymers of polylysine andPEG; polyethylene imine; copolymers of polyethylene imine and PEG;polyhydroxyacids; polyanhydrides; polyhydroxyalkanoates,poly(L-lactide-co-L-lysine); poly(serine ester);poly(4-hydroxy-L-proline ester); poly-α-(4-aminobutyl)-L-glycolic acid;derivatives thereof; combinations thereof; and copolymers thereof.

Further examples of polymeric and non-polymeric materials that can beused in particles of several embodiments include, but are not limitedto, poly(lactide), poly(hydroxybutyrate), poly(beta-amino) esters and/orcopolymers thereof. Alternatively, the particles can comprise othermaterials, including but not limited to, poly(dienes) such aspoly(butadiene) and the like; poly(alkenes) such as polyethylene,polypropylene and the like; poly(acrylics) such as poly(acrylic acid)and the like; poly(methacrylics) such as poly(methyl methacrylate),poly(hydroxyethyl methacrylate), and the like; poly(vinyl ethers);poly(vinyl alcohols); poly(vinyl ketones); poly(vinyl halides) such aspoly(vinyl chloride) and the like; poly(vinyl nitriles), poly(vinylesters) such as poly(vinyl acetate) and the like; poly(vinyl pyridines)such as poly(2-vinyl pyridine), poly(5-methyl-2-vinyl pyridine) and thelike; poly(styrenes); poly(carbonates); poly(esters); poly(orthoesters);poly(esteramides); poly(anhydrides); poly(urethanes); poly(amides);cellulose ethers such as methyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose and the like; cellulose esters such ascellulose acetate, cellulose acetate phthalate, cellulose acetatebutyrate; and polysaccharides. These materials may be used alone, asphysical mixtures (blends), or as copolymers.

In several embodiments, a particle comprises a semiconductornanocrystal. A semiconductor nanocrystal is a nanocrystal of Group II-VIand/or Group III-V semiconductor compounds. Examples of semiconductornanocyrstals include, but are not limited to Group II-VI semiconductorssuch as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe,BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well asmixed compositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof.

In several embodiments, a particle comprises a metal particle, such asan Au, Ag, Pd, Pt, Cu, Ni, Co, Fe (e.g. iron sulfide), Mn, Ru, Rh, Os,or Ir particle. In various embodiments, a particle comprises a metaloxide particle. Examples of suitable metal oxide particles include zincoxide, titanium (di)oxide, iron oxide, silver oxide, copper oxide,aluminum oxide, or silicon (di)oxide particles. In certain embodiments,a particle comprises a magnetic particle, such as a magnetic bead,nanoparticle, microparticle, and the like.

In some embodiments, a particle can be associated with a capture probe,such as a second capture probe. In some embodiments, the capture probecan be attached to the particle. In some embodiments, the capture probecomprises a catalyst. In some embodiments, the catalyst compriseshorseradish peroxidase. In some embodiments, the capture probe comprisesa plurality of catalysts. In some embodiments, the catalysts are thesame. In some embodiments, one or more catalysts are different.

In some embodiments, a particle can be associated with a catalyst. Insome embodiments, the catalyst can be attached to the particle. In someembodiments, the catalyst comprises horseradish peroxidase. In someembodiments, the particle comprises a plurality of catalysts. In someembodiments, the catalysts are the same. In some embodiments, one ormore catalysts are different.

Catalysts

Some embodiments of the methods, compositions and systems providedherein include catalysts. In some embodiments, the presence of acatalyst is useful to amplify a signal. In some embodiments, a catalystpromotes a reaction that results in the formation of a precipitate. Insome embodiments, a catalyst comprises an enzyme. Examples of suchenzymes include horseradish peroxidase, alkaline phosphatase, andβ-galactosidase. Examples of reagents useful with such catalysts include4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole.

In some embodiments, a catalyst is attached to a capture probe. In someembodiments, a catalyst is attached to a capture probe directly. In someembodiments, a capture probe comprises a first affinity tag, and acatalyst comprises a second affinity tag. In some such embodiments, thefirst affinity tag can bind to the second affinity tag, thereforeassociating the capture probe and catalyst with each other. Examples ofaffinity tags include biotin, streptavidin, poly-His, and nickel, andderivatives thereof.

Methods for Measuring a Target Analyte

Some embodiments of the methods, compositions and systems providedherein include a method for detecting and/or measuring the level of atarget analyte. Some such embodiments include obtaining a planarsubstrate comprising an optical sensor having a first capture probeattached thereto; contacting the first capture probe with a samplecomprising a target analyte that selectively binds to the first captureprobe; contacting the bound target analyte with a second capture probethat selectively binds to a complex comprising the bound target analyte,wherein the second capture probe comprises a catalyst; contacting thecatalyst with a reagent under conditions where the reagent forms aprecipitate in the presence of the catalyst; and measuring a change inresonance wavelengths at the optical sensor, thereby measuring the levelof the target analyte.

In some embodiments, a method of detecting a target analyte includesobtaining a planar substrate comprising an optical sensor having a firstcapture probe attached thereto; contacting the first capture probe witha sample comprising a target analyte that selectively binds to the firstcapture probe; contacting the bound target analyte with a second captureprobe that selectively binds to a complex comprising the bound targetanalyte, wherein the second capture probe comprises a first affinitytag; contacting the bound second capture probe with a second affinitytag that selectively binds to the bound first affinity tag, wherein thesecond affinity tag comprises a catalyst; contacting the catalyst with areagent under conditions where the reagent forms a precipitate in thepresence of the catalyst; and measuring an increase in precipitateformation at the optical sensor, thereby detecting the target analyte.In some embodiments, an increase in precipitate formation is indicativeof the level of the target analyte in the sample. In some embodiments,the quantity of precipitate formation is indicative of the level of thetarget analyte in the sample. In some embodiments, an increase in therate of precipitate formation is indicative of the level of targetanalyte in the sample. In some embodiments, an increase in inprecipitate formation is measured by determining a change in resonancewavelengths at the optical sensor.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe selectively binds to thetarget analyte. In some embodiments, the second capture probeselectively binds to the bound target analyte. In some embodiments, thesecond capture probe comprises an antibody or antigen-binding fragmentthereof. In some embodiments, the second capture probe comprises anantibody selected from the group consisting of, anti-IL-2 from clone555051, anti-IL-2 from clone 555040, anti-IL-2 from clone MQ1-17H12,anti-IL-6 from clone BAF206, anti-IL-6 from clone MAB206, anti-IL-6 fromclone MQ2-13A5, anti-IL-6 from clone MQ2-39C3, and an antigen-bindingfragment thereof.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Insome embodiments, the reagent is selected from the group consisting of4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole.

In some embodiments, the catalyst is contacted with an additional agent,such as hydrogen peroxide. In some embodiments, the concentration ofhydrogen peroxide is less than about 0.001%, 0.002%, 0.003%, 0.002%,0.001%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, and 0.0003%, orwithin a range between any two of the foregoing concentrations.

In some embodiments, the optical sensor is washed between any of theforegoing steps.

In some embodiments, a target analyte is detected having a concentrationless than about 1000 pg·ml, 500 pg/ml, 100 pg/ml, 50 pg/ml, 10 pg/ml,and 1 pg/ml, or within a range between any two of the foregoingconcentrations.

In some embodiments, the target analyte comprises a cytokine. In someembodiments, the analyte is selected from the group consisting of IL-2,IL-4, IL-6, and IL-8.

In some embodiments, a sample comprises the analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator. In some embodiments, the optical sensor comprises a waveguidestructure. In some embodiments, the optical sensor comprises a well. Insome embodiments, the planar substrate comprises a plurality of opticalsensors. In some embodiments, a planar substrate can include at leastabout 1, 2, 5, 10, 100, 1000, 10000, 100000, 1000000 optical sensors, ora planar substrate can include a plurality of optical sensors, in whichthe plurality is a number within a range between any of the foregoingnumbers of optical sensors. In some embodiments, the planar substratecomprises a thermal control. In some embodiments, the planar substratecomprises an optical chip. In some embodiments, the planar substratecomprises a multiwell plate. In some embodiments, the planar substratecomprises a flowcell.

Kits

Some embodiments of the methods, compositions and systems providedherein include a kit for measuring the level of a target analyte. Somesuch kits include a planar substrate comprising an optical sensor havinga first capture probe attached thereto, wherein the first capture probeselectively binds to the target analyte; a second capture probe thatselectively binds to a complex comprising the target analyte bound tothe first capture probe, wherein the second capture probe comprises acatalyst; and a reagent that can form a precipitate in the presence ofthe catalyst. In some embodiments, the second capture probe comprises afirst affinity tag. In some embodiments, a second affinity tag thatselectively binds to the first affinity tag, wherein the second affinitytag comprises the catalyst.

In some embodiments, a kit for detecting a target analyte comprising: aplanar substrate comprising an optical sensor having a first captureprobe attached thereto, wherein the first capture probe selectivelybinds to the target analyte; a second capture probe that selectivelybinds to a complex comprising the target analyte bound to the firstcapture probe, wherein the second capture probe comprises a firstaffinity tag; a second affinity tag that selectively binds to the firstaffinity tag, wherein the second affinity tag comprises a catalyst; areagent that can form a precipitate in the presence of the catalyst.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe selectively binds to thetarget analyte. In some embodiments, the second capture probeselectively binds to the bound target analyte. In some embodiments, thesecond capture probe comprises an antibody or antigen-binding fragmentthereof. In some embodiments, the second capture probe comprises anantibody selected from the group consisting of, anti-IL-2 from clone555051, anti-IL-2 from clone 555040, anti-IL-2 from clone MQ1-17H12,anti-IL-6 from clone BAF206, anti-IL-6 from clone MAB206, anti-IL-6 fromclone MQ2-13A5, anti-IL-6 from clone MQ2-39C3, and an antigen-bindingfragment thereof.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Insome embodiments, the reagent is selected from the group consisting of4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole.

In some embodiments, the kit includes hydrogen peroxide. In someembodiments, the concentration of hydrogen peroxide is less than about0.001%, 0.002%, 0.003%, 0.002%, 0.001%, 0.009%, 0.008%, 0.007%, 0.006%,0.005%, 0.004%, and 0.0003%, or within a range between any two of theforegoing concentrations.

In some embodiments, the kit is adapted to detect a target analyte isdetected having a concentration less than about 1000 pg·ml, 500 pg/ml,100 pg/ml, 50 pg/ml, 10 pg/ml, and 1 pg/ml, or within a range betweenany two of the foregoing concentrations.

In some embodiments, the kit is adapted to detect a target analytecomprising a cytokine. In some such embodiments, the analyte is selectedfrom the group consisting of IL-2, IL-4, IL-6, and IL-8.

In some embodiments, a sample comprises the analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator. In some embodiments, the optical sensor comprises a waveguidestructure. In some embodiments, the optical sensor comprises a well. Insome embodiments, the planar substrate comprises a plurality of opticalsensors. In some embodiments, a planar substrate can include at leastabout 1, 2, 5, 10, 100, 1000, 10000, 100000, 1000000 optical sensors, ora planar substrate can include a plurality of optical sensors, in whichthe plurality is a number within a range between any of the foregoingnumbers of optical sensors. In some embodiments, the planar substratecomprises a thermal actuator. In some embodiments, the planar substratecomprises an optical chip. In some embodiments, the planar substratecomprises a multiwell plate. In some embodiments, the planar substratecomprises a flowcell.

Systems

Some embodiments of the methods, compositions and systems providedherein include a system for measuring the level of a target analytecomprising. Some such systems include: a planar substrate comprising anoptical sensor having a first capture probe attached thereto, whereinthe target analyte selectively binds to the first capture probe; asecond capture probe that selectively binds to a complex comprising thetarget analyte bound to the first capture probe, wherein the secondcapture probe comprises a catalyst; a reagent which can form aprecipitate in the presence of the catalyst; and a detector adapted tomeasure a change in resonance wavelengths at the optical sensor. In someembodiments, a change in resonance wavelengths at the optical sensor isindicative of the level of the target analyte. In some embodiments, thesecond capture probe is formed by contacting an affinity molecule boundto a first affinity tag with a second affinity tag bound to a catalyst,wherein the second affinity tag selectively binds to the first affinitytag. In some embodiments, the second capture probe comprises an affinitytag bound to a catalyst.

In some embodiments, a system of detecting a target analyte comprises: aplanar substrate comprising an optical sensor having a first captureprobe attached thereto, wherein the target analyte selectively binds tothe first capture probe; a second capture probe that selectively bindsto a complex comprising the target analyte bound to the first captureprobe, wherein the second capture probe comprises a first affinity tag;a second affinity tag that selectively binds to the bound first affinitytag, wherein the second affinity tag comprises a catalyst; a reagentthat forms a precipitate in the presence of the catalyst; and a detectoradapted to measure an increase in precipitate formation.

In some embodiments, the increase in precipitate formation is indicativeof the level of the target analyte in the sample.

In some embodiments, the first and second affinity tags are eachselected from the group consisting of biotin, streptavidin, poly-His,and nickel.

In some embodiments, an increase in precipitate formation is measured bya change in resonance wavelengths at the optical sensor.

In some embodiments, the first capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the first captureprobe comprises an antibody selected from the group consisting ofanti-IL-2 from clone 555051, anti-IL-2 from clone 555040, anti-IL-2 fromclone MQ1-17H12, anti-IL-6 from clone BAF206, anti-IL-6 from cloneMAB206, anti-IL-6 from clone MQ2-13A5, anti-IL-6 from clone MQ2-39C3,and an antigen-binding fragment thereof.

In some embodiments, the second capture probe comprises an antibody orantigen-binding fragment thereof. In some embodiments, the secondcapture probe selectively binds to the target analyte. In someembodiments, the second capture probe selectively binds to the boundtarget analyte. In some embodiments, the second capture probe comprisesan antibody selected from the group consisting of, anti-IL-2 from clone555051, anti-IL-2 from clone 555040, anti-IL-2 from clone MQ1-17H12,anti-IL-6 from clone BAF206, anti-IL-6 from clone MAB206, anti-IL-6 fromclone MQ2-13A5, anti-IL-6 from clone MQ2-39C3, and an antigen-bindingfragment thereof.

In some embodiments, the catalyst is selected from the group consistingof horseradish peroxidase, alkaline phosphatase, and β-galactosidase. Insome embodiments, the reagent is selected from the group consisting of4-chloro-1-naphthol, Hanker-Yates reagent, 3,3′-diaminobenzidine, and3-amino-9-ethylcarbazole.

In some embodiments, an additional reagent includes hydrogen peroxide.In some embodiments, the concentration of hydrogen peroxide is less thanabout 0.001%, 0.002%, 0.003%, 0.002%, 0.001%, 0.009%, 0.008%, 0.007%,0.006%, 0.005%, 0.004%, and 0.0003%, or within a range between any twoof the foregoing concentrations.

In some embodiments, the planar surface is adapted for washing theoptical sensor.

In some embodiments, the system is adapted to detect a target analyte isdetected having a concentration less than about 1000 pg·ml, 500 pg/ml,100 pg/ml, 50 pg/ml, 10 pg/ml, and 1 pg/ml, or within a range betweenany two of the foregoing concentrations. In some embodiments, theanalyte comprises a cytokine. In some embodiments, the analyte isselected from the group consisting of IL-2, IL-4, IL-6, and IL-8.

In some embodiments, a sample comprises the analyte. In someembodiments, the sample is selected from the group consisting of serumand cerebrospinal fluid.

In some embodiments, the optical sensor comprises an optical ringresonator. In some embodiments, the optical sensor comprises a waveguidestructure. In some embodiments, the optical sensor comprises a well. Insome embodiments, the planar substrate comprises a plurality of opticalsensors. In some embodiments, a planar substrate can include at leastabout 1, 2, 5, 10, 100, 1000, 10000, 100000, 1000000 optical sensors, ora planar substrate can include a plurality of optical sensors, in whichthe plurality is a number within a range between any of the foregoingnumbers of optical sensors. In some embodiments, the planar substratecomprises a thermal control. In some embodiments, the planar substratecomprises an optical chip. In some embodiments, the planar substratecomprises a multiwell plate. In some embodiments, the planar substratecomprises a flowcell.

EXAMPLES Example 1—IL-2 and IL-4 Assays

This example illustrates development of an assay for detection ofexample target analytes, IL-2 and IL-4, using enzymatic enhancement of asandwich based assay with microring detection. This overall assay isdepicted schematically in FIG. 5. Briefly, in a four-step assay, acytokine sandwich assay with a biotinylated secondary antibody isfollowed by addition of streptavidin-HRP conjugate. Subsequentintroduction of freshly prepared hydrogen peroxide in 4-Cl-1-naphtholsubstrate solution allows for the catalytic formation of the4-C1-1-naphthon precipitate only at rings with bound HRP.

Optimization of Enzymatic HRP Process for Rapid Precipitate Formation

Sandwich assays and antibody pair selections have been demonstrated inprevious work with IL-2^(9, 11) and IL-6¹⁰. Due to extensive work withthese targets in previous work, the advances described in thisdisclosure have a well-defined benchmark for assay sensitivitycomparisons and assay complexity and time-to-result tradeoffs. Thoughenzymatic amplification introduces complexity and extra time to theassay, the impressive sensitivity gains and highly quantitative signalsmake the assay extremely valuable for expanding the potentialapplications for ring resonator biosensing.

Selection of 4-Cl-1-Naphthol as HRP Substrate

HRP is a commonly used enzymatic label that catalyzes decomposition oftwo molecules of its natural substrate, hydrogen peroxide, into waterand oxygen.¹² Since HRP has low selectivity for its natural substrate,many chromogenic substrates have been developed and compared for use asHRP substrates with ELISA.¹³ These include 4-chloro-1-naphthol (4-CN),o-phenylenediamine, 3-amino-9-ethyl carbazole,2,2′-azino-bis(3-ethylbenzthiazolone), 4-aminoantipyrene+a phenol,3,3′-diaminobenzidine (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB),2,2′-azino-bis(3-ethylbenzthiazolone), Hanker-Yates reagent (HYR),3-methyl-2-benzothiazolinone hydrazone hydrochloride(MBTH)+3-dimethylaminobenzoic acid, o-dianisidine, and dicarboxidine. Ofthese possible substrates and others, only some produce water-insolubleproducts following oxidation: 4-CN, HYR, DAB, and3-amino-9-ethylcarbazole.¹² HRP is known to catalyze the oxidation ofthe substrate 4-CN by hydrogen peroxide to form the insoluble blueprecipitate, 4-Cl-1-naphthon.¹⁴ Oxidation of the 4-CN substrate produces4-Cl-1-naphthon, a water-insoluble blue precipitate. Hydrogen peroxideoxidizes 4-CN, albeit very slowly, without HRP. Thus, it is preferableto combine the substrate and hydrogen peroxide immediately before thefinal step of the microring sandwich assay. Streptavidin-HRP conjugatesbound to biotinylated secondary antibodies catalyze 4-Cl-1-naphthonprecipitate formation at microrings that have bound the target analyte.This reaction scheme is presented in FIG. 4. Though traditional ELISAmakes use of the blue color change following substrate oxidation, therequirements of a RI-based ring resonator assay require differentoptimization. Color intensity and resistance to fading is irrelevant tothis work, as the signal arises simply from precipitation of theoxidized substrate on specific microrings. Relevant factors includeprecipitation rate, precipitate deposition specificity, andreproducibility of the reaction. TMB was also evaluated as an HRPsubstrate without success. Though blue color was formed as evidence ofthe enzymatic process, the TMB did not effectively precipitate on therings to amplify the signal. TMB seems to have poor solubilitycharacteristics, despite its use in SPRI amplification.⁸

Materials

4-CN solution (0.48 mM 4-Chloro-1-naphthol in 50 mM Tris-HCl/0.2 MNaCl/17% methanol), a product normally used for Western Blotvisualization, was obtained from Sigma-Aldrich (St. Louis, Mo.).High-sensitivity streptavidin-HRP (SA-HRP) conjugate and EZ-LinkNHS-PEG4-Biotin were obtained from Pierce (Rockford, Ill.), and 30%hydrogen peroxide was obtained from Macron Chemicals (Center Valley,Pa.). anti-IL-2 clone 555051 (capture Ab) and biotin anti-IL-2 clone555040 (detection Ab) were obtained from BD Biosciences (San Jose,Calif.). anti-IL-2 43-2 and HRP-conjugated anti-IL-2 14-0 were obtainedfrom Abcam (Cambridge, Mass.), while anti-IL-2 MQ1-17H12, polyclonalanti-IL-2, and recombinant human IL-2 were obtained from eBioscience(San Diego, Calif.). anti-IL-6 clone MAB206 (capture Ab) and biotinanti-IL-6 clone BAF206 (detection Ab) were obtained from R&D Systems(Minneapolis, Minn.). Recombinant human IL-6, anti-IL-6 clone MQ2-13A5,and anti-IL-6 clone MQ2-39C3 were obtained from eBioscience (San Diego,Calif.). Mouse IgG1k isotype control clone P3 and anti-IL-4 clone 8D4-8(both from eBioscience) were used as control antibodies to verify assayselectivity.

Antibody Functionalization Methods

Ring resonator optical scanning instrumentation, software, and chipshave been described previously.^(15, 16) Prior to functionalization,chips were cleaned by a 30-s immersion in piranha solution (3:1H₂SO₄:30% H₂O₂) followed by a water rinse and drying in a nitrogenstream. After a 10-min sonication in 100% ethanol, chips were dried in anitrogen stream and spotted with 30 μL of 1 mg/mL HyNic silane(3-N-((6-(N′-isopropylidene-hydrazino))nicotinamide)propylthriethoxysilane,SoluLink, San Diego, Calif.) in 95% ethanol/5% dimethylformamide (DMF)for a 20-min incubation at room temperature to install a hydrazinemoiety. Following silanization, chips were sonicated for 5 min in 100%ethanol to remove physisorbed silane. After drying in a nitrogen stream,chips were loaded into a previously described fluidic cell¹⁶ with acustom 4-channel fluidic gasket¹⁷ (Scarpati Technical Services/RMSLaser, San Diego, Calif.) to direct antibody solution flow to definedgroups of 4-6 rings each.

Capture antibodies were first buffer-exchanged into 100 mMphosphate-buffered saline (PBS) pH 7.4 with spin desalting columns (7 kmolecular weight cutoff, Pierce). In separate reaction vials, lysineresidues on the 4 capture antibodies were functionalized with aldehydemoieties by reacting 0.5 mg/mL antibody with a 10-fold molar excess ofsuccinimidyl-4-formyl benzoate (S-4FB, SoluLink, 0.5 mg/mL in DMF) for 2h at room temperature. After another buffer exchange into 100 mM PBS pH7.4 to remove excess S-4FB and DMF, the antibody solutions were dilutedto 10 μg/mL in 100 mM PBS pH 6 with 50 mM aniline. The captureantibody-4FB conjugates were flowed over the chip using the previouslydescribed fluidics controlled by a multi-channel programmable syringepump (BS-9000-8, Braintree Scientific Inc., Braintree, Mass.) operatedin withdraw mode at 2 μL/min. Covalent antibody attachment by hydrazonebond formation between the hydrazine silane surface andaldehyde-modified antibodies was catalyzed by aniline in the runningbuffer to allow full surface coverage after 30-60 min. Four-channelantibody functionalization was performed such that each of the twochannels of 12 sensor microrings had anti-IL-2 oranti-IL-6-functionalized rings upstream of control (anti-IL-4 of mouseIgG isotype control) rings, as described in FIG. 6. Groups of four tosix rings in each of four channels were functionalized simultaneously.This functionalization strategy allowed anti-IL-2 rings to be upstreamof anti-IL-4 control rings when fluidics are switched to a two-channelsetup after the blocking step (FIG. 6). After multiplexed antibodyfunctionalization, chips were blocked overnight at 4° C. inStartingBlock PBS buffer (cat. #37538, Pierce).

Optimized Assay Conditions

For enzymatic amplification of cytokine sandwich assays, a four-stepassay was designed. This assay is depicted schematically in FIG. 5.After primary binding of cytokines to anti-cytokine capture antibodieson the rings, secondary biotin anti-cytokine antibodies were introducedto form a sandwich pair. Next, complexation of streptavidin-HRP (SA-HRP)conjugate to the sandwich pair introduces the enzyme at appropriaterings in a concentration-dependent manner. Finally flowing hydrogenperoxide with 4-CN causes specific precipitation of 4-Cl-1-naphthon atthe ring surface. The assay conditions (75-min total assay time,including rinse steps) are as follows (NOTE: all steps maintain a 30μL/min flow rate and utilize room-temperature reagents):

-   -   (1) 20-min primary cytokine binding by incubation with sample or        standard in 10 mM PBS pH 7.4+0.1 mg/mL BSA+0.05% Tween-20        (PBS-BSA-T);    -   (2) 2-min buffer rinse (PBS-BSA-T);    -   (3) 10-min binding of biotinylated secondary anti-cytokine        antibody (2 μg/mL in PB S-BSA-T);    -   (4) 2-min buffer rinse (PBS-BSA-T);    -   (5) 10-min binding of streptavidin-HRP conjugate (2 μg/mL in        PBS-BSA-T);    -   (6) 3-min buffer rinse (PBS-BSA-T);    -   (7) 25-min introduction of 465 μM 4-CN/0.01% hydrogen peroxide        solution in 17% methanol for precipitation of 4-Cl-1-naphthon;    -   (8) Buffer rinse (PBS-BSA-T).

The ratio of 4-CN to hydrogen peroxide was carefully selected foroptimal signal amplification and precipitation rate; other ratios of4-CN:hydrogen peroxide gave little to no signal, and excess hydrogenperoxide was observed to quench the reaction. Preferably, this solutionwas prepared directly (within 1-2 min) before use by adding 45 μL of0.3% hydrogen peroxide (1:100 dilution of 30% hydrogen peroxide indistilled water) to 1.5 mL of 480 μM 4-CN solution in methanol (fromSigma-Aldrich) that has been already brought to room temperature. Thesolution was vortexed to mix and then immediately used in Step 7 above.The 17% methanol in the 4-CN solution caused a ˜300-pm bulk RI shift,immediately followed by specific precipitation on appropriate rings in acytokine-concentration-dependent manner. A fast flow rate (30 uL/min)was necessary to ensure that substantial uncatalyzed reaction of 4-CNand hydrogen peroxide did not occur within the inlet tubing prior toreaching the chip.

Enzymatic Amplification of IL-2 Sandwich Assay

The first step in creating an enzymatic amplification assay for IL-2involved screening a variety of antibody pairs to determine the optimalcapture and detection antibody combination. FIG. 7 shows a graph for anassay to screen IL-2 antibody sandwich pairs in which 10 ng/mL IL-2sandwich assays were performed with a variety of antibody sandwich pairsinvolving either clone 555051 (BD) or clone 43-2 (Abcam) as captureantibody. In each case, primary 10 ng/mL IL-2 binding was evident on555051, but not on 43-2. Only 555051 was a suitable capture antibody forIL-2, and biotin anti-IL-2 555040 (BD) represents the most effectivedetection antibody (MQ1-17H12 from eBioscience also demonstratedeffectiveness as a detection antibody). A low-pH rinse was used toregenerate the capture antibodies between each sandwich assay test. Allresponses are corrected to anti-IL-5 control rings. As shown in FIG. 7,capture antibody clone 555051 paired with detection antibody 555040shows the greatest affinity and signal response in the 10 ng/mL IL-2test. This result highlights the use of comparing commercial antibodiesside-by-side using an identical assay on an identical biosensingplatform.

With the highest affinity antibody pair chosen, the next step wasoptimization of enzymatic amplification assay conditions. Initialattempts with 1) TMB as an HRP substrate and 2) with diluteconcentrations of 4-CN substrate solution were unsuccessful. Since 4-CNis provided in a 17% methanol solution, initial work sought to preventthe bulk RI shift associated with high solvent concentration bysubstantially diluting this methanol solution. An example of anenzymatically amplified IL-2 sandwich assay under dilute 4-CN andhydrogen peroxide conditions is shown in FIG. 8. In the assay depictedin FIG. 8, 100 pg/mL IL-2 sandwich assay with enzymatic amplificationwas carried out under dilute 4-CN/peroxide conditions. Though clearlydetectable upon addition of streptavidin-HRP conjugate (SA-HRP), theaddition of dilute 4-CN and peroxide provided only minor signal boost.Interestingly, despite no measurable response to the IL-2 sandwich onthe clone 43-2 rings during the first three assay steps, the finalamplification step gave a small signal. All responses were corrected toanti-IL-5 control rings. Though 100 pg/mL IL-2 was clearly detectedbefore the fourth assay step (addition of 4-CN and peroxide), the finalenzymatic amplification step provided only a minor signal boost.Furthermore, it was also found that adding additional hydrogen peroxidedid not improve the enzymatic deposition of 4-Cl-1-naphthon.

FIG. 9 depicts quenching of enzymatic deposition of 4-Cl-1-naphthonprecipitate by high hydrogen peroxide concentration in which after avariety of 10 ng/mL IL-2 sandwich assay antibody combinations (whichconfirm and extend the data in FIG. 7), enzymatic amplification wascarried out with streptavidin-HRP conjugate (SA-HRP) at t=160 minfollowed by dilute 4-CN/hydrogen peroxide at t=173 min. The initial 48μM 4-CN/0.0003% hydrogen peroxide solution was spiked with 0.3% hydrogenperoxide at t=186 min to a final concentration of 0.003% hydrogenperoxide, which appeared to quench the precipitation reaction. Thisprocess was carried out at a low flow rate (5 μL/min). Importantly, noprecipitation was observed on control anti-IL-4 rings. As shown in FIG.9, increasing the peroxide concentration from 0.0003% (100 μM) to 0.003%(1 mM) while maintaining dilute (48 μM) 4-CN appeared to quench theenzymatic process. This result demonstrates the narrow window of4-CN:H₂O₂ ratio that is required for effective enzymatic deposition of4-Cl-1-naphthon.

Further testing of reaction conditions demonstrated that a combinationof more concentrated 4-CN substrate coupled with a higher flow rate arevital to efficient precipitate deposition. The final assay step involves465 μM 4-CN and 0.01% (3 mM) H₂O₂ at 30 μL/min.

FIG. 10 depicts that large signal amplification with enzymaticamplification gives nm-scale shifts down to 100 pg/mL IL-2 in whichrepresentative rings for each of three IL-2 concentrations showed aconcentration-dependent resonant wavelength shift upon addition of 465μM 4-CN and 0.01% H₂O₂ to bound streptavidin-HRP conjugate (SA-HRP) att=50 min. The inset of FIG. 10 shows a zoom into the region of t=0-48min prior to enzymatic amplification, demonstrating secondary antibodyand SA-HRP shifts that are evident at 1 and 10 ng/mL IL-2concentrations. The relative shift axis for the zoomed-in region isshown to the right of the inset of FIG. 10. These optimized assayconditions were used in subsequent IL-2 calibration experiments. Asshown in FIG. 10, nm-scale resonant wavelength shifts are observed underoptimized 4-CN and H₂O₂ conditions. 100 pg/mL represents the lowestconcentration ever detected with a traditional IL-2 ring resonatorsandwich assay (<1 pm shift), but this concentration generates a nearly1 nm shift with enzymatic amplification. Following an initial ˜300-pmbulk RI shift upon addition of substrate (due to 17% methanol in the4-CN solution), 4-Cl-1-naphthon precipitates specifically on anti-IL-2rings. With these conditions, a large-scale calibration of IL-2enzymatic response was performed next.

As described in FIG. 6, the chips were functionalized so as to enabletwo independent sets of anti-IL-2 capture antibody rings upstream ofanti-IL-4 control rings. Thus, each chip was simultaneously used for twoIL-2 concentration standards in independent fluidic channels. FIG. 11shows the result of the multi-chip calibration of enzymatic signal forIL-2 sandwich assays (concentration range: 1 pg/mL-1 ng/mL). In theassay depicted in FIG. 11, IL-2 calibration with four-step HRP enzymaticamplification assay. The addition of IL-2 (t=3 min), biotin anti-IL-2555040 (t=25 min), streptavidin-HRP (t=37 min), 465 μM 4-CN/0.01% H₂O₂(t=50 min), and a final buffer rinse (t=75 min) are marked by the dashedlines. Based on two-channel multi-chip calibration at a variety of IL-2concentrations, FIG. 11 demonstrates the ability to quantify IL-2 over abroad dynamic range with a detection limit of 1 pg/mL using a ˜1-hassay. Concentration-dependent responses were observed over a rangespanning 4 logs (1-10,000 pg/mL, see FIG. 10 for 10,000 pg/mL response).All responses were corrected to downstream anti-IL-4-functionalizedrings, which were necessary for subtracting off the ˜300-pm bulk RIshift at t=50 min due to 17% methanol in the 4-CN solution. Thiscalibration plot shows that IL-2 concentrations as low as 1 pg/mL can bedetected using enzymatic amplification, although 1 and 2 pg/mL signalsare difficult to differentiate in this case. However, substantialdifferentiation of 5 and 10 pg/mL standards shows that the assay isideally suited for samples containing cytokine concentrations between 5and 1000 pg/mL. This concentration range enables significant broadeningof the range of applications for cytokine analysis on the ring resonatorplatform. Importantly, all responses are corrected to downstreamanti-IL-4-functionalized rings, which was necessary for subtracting offthe ˜300-pm bulk RI shift at t=50 min due to 17% methanol in the 4-CNsolution. Negative control experiments (0 ng/mL) showed no enzymaticsignal, demonstrating a low background (data not shown). Though IL-2quantitation with the four-step HRP enzymatic amplification assayrequires extra time, the total assay time of just over 1 h is stillreasonable and superior to the ELISA time-to-result. By combining thedata shown in FIGS. 10 and 11, concentration-dependent enzymaticprecipitation responses were observed over a range spanning four logs(1-10,000 pg/mL). To verify the effectiveness of the assay for anadditional target, a similar assay was also designed for the cytokineinterleukin-6 (IL-6).

Enzymatic Amplification of IL-6 Sandwich Assay

The same enzymatic amplification assay was applied to IL-6 detection.Capture anti-IL-6 clone MAB206 and biotinylated detection anti-IL-6clone BAF206 (both from R&D Systems) were chosen for initialcharacterization of the assay, although similar results were alsoobtained for clone MQ2-13A5 as capture antibody. An example of IL-6enzymatic amplification is shown in FIG. 12, displaying the signals for5 and 50 pg/mL IL-6. In FIG. 12, the addition of IL-6 (t=3 min), biotinanti-IL-6 BAF206 (t=25 min), streptavidin-HRP (t=37 min), 465 μM4-CN/0.01% H₂O₂ (t=50 min), and a final buffer rinse (t=75 min) aremarked by the dashed lines. The capture antibody is anti-IL-6 cloneMAB206. All responses were corrected to downstream mouse IgG isotypecontrol antibody-functionalized rings, which were preferred forsubtracting off the ˜300-pm bulk RI shift at t=50 min due to 17%methanol in the 4-CN solution. The magnitudes of the shifts at the twoIL-6 concentrations were comparable to the IL-2 responses at similarconcentrations (FIG. 11). The IL-2 antibody pair showed slightly betteraffinity for IL-2 than the IL-6 antibody pair for IL-6. IL-6 detectiondown to 5 pg/mL was possible, but further work may permit detection downto 1 pg/mL as was achieved with IL-2.

CONCLUSIONS

In this Example, an optimized HRP-catalyzed amplification scheme wassuccessful for achieving unprecedented cytokine detection limits on thering resonator biosensing platform. In the past, traditional cytokinesandwich assays on ring resonators were quantitative down to 100 pg/mL-1ng/mL, depending on the particular analyte and assay format. Thisdisclosure shows that HRP-catalyzed amplification of the cytokinesandwich assay provides a full two orders of magnitude improvement inthe detection limit for IL-2. The 1 pg/mL (65 fM) limit of detectionrepresents the lowest concentration of any analyte detected with ringresonators to date. Importantly, the enzymatic process displays a lowerand more reproducible background than the bead-based assay, alsoremoving the need for time-consuming bead exchange directly beforerunning the assay.¹⁰ By providing detection capabilities throughout thepg/mL regime, this assay will allow applications that go beyond cellculture work at high cell densities. The ability to quantitate cytokinesin the 1-100 pg/mL range is useful for many challenging proteinbiosensing applications, including serum and cerebrospinal fluid (CSF)diagnostics.

Initial work with IL-6, which has displayed assay performance similar toIL-2, opens up the opportunity to perform CSF diagnostics with apossible Alzheimer's Disease (AD) biomarker. IL-6 is a part of the acuteinflammatory response, produced by macrophages and monocytes at areas ofinflammation.¹⁹ IL-6, originally known as interferon-beta₂, acts as thechief stimulator for production of most acute phase proteins (C-reactiveprotein, factor B complement, C3, etc).²⁰ As a general marker ofinflammation, IL-6 is released from contracting skeletal muscles intothe blood plasma during exercise without altering CSF levels of IL-6,signifying a separation of the CSF and plasma pools of IL-6.²¹ Outsideof exercise-induced increases in plasma IL-6 levels, IL-6 concentrationchanges in CSF have been associated with brain trauma,²² stroke,²³multiple sclerosis,²⁴ post-traumatic stress disorder,²⁵ assortedinfections of the central nervous system,²⁶ and AD.²⁷ Thesestudies,²²⁻²⁷ which use commercial ELISAs to compare a variety ofdisease states to healthy controls, report healthy IL-6 CSF levels inthe 1-20 pg/mL range. In general, IL-6 levels noted in thesestudies²²⁻²⁷ are observed to increase in CSF as well as plasma or serum,with elevated IL-6 levels often reaching 100-1000 pg/mL. Though a numberof studies have looked at AD-induced changes in CSF concentrations ofIL-6 and other cytokines,²⁸ the effect of AD progression on IL-6 levelsremains unclear.²⁷ Discrepancies and inconsistencies exist among avariety of studies aimed at evaluating changes in IL-6 levels associatedwith AD: IL-6 levels have been reported to increase²⁹⁻³¹ or to notchange significantly in AD.^(28, 32-34) Since IL-6 has definitively beenobserved by immunohistochemistry in the brain plaques of AD patients³⁵and is present at significantly higher levels in AD brain tissuehomogenates,³⁶ it is possible (or even likely) that the discrepancies inIL-6 expression alterations in AD CSF are due to assay inaccuracy andimprecision that cause a lack of statistical significance.

Example 2—IL-2, IL-6 and IL-8 Assays

This example illustrates detection and quantitation of target analytes,IL-2, IL-6, and IL-8, and detection of target analytes in a biologicalsample, cerebrospinal fluid. The overall strategy for the HRP-enhanceddetection of protein targets is shown in FIG. 5. This strategy providesa quantitative method for increasing the per-analyte sensor response.

Materials: PBS buffer was reconstituted from Dulbecco's phosphatebuffered saline packets purchased from Sigma-Aldrich (St. Louis, Mo.)into two formulations. A PBS buffer for antibody functionalization (PBS6) was reconstituted at 10× concentration with the addition of 50 mManiline and adjusted to pH 6.0. A PBS running buffer (PBS 7.4) wasreconstituted at 1× concentration with the addition of 0.1 mg/mL BSA atadjusted to pH 7.4. Starting Block was purchased from Thermo Scientificand used as a blocking agent to prevent nonspecific fouling of thesensor surface. The silane3-N-((6-(N′-isopropylidene-hydroazino))nicotinamide)propyltriethyoxysilane(HyNic Silane) and succinimidyl-4-formylbenzamide (S-4FB) were purchasedfrom Solulink. Protein desalting columns were purchased from ThermoScientific. Recombinant interleukin proteins, antibodies, and ELISA kitswere obtained from eBioscience, BD Biosciences, or R&D Systems.Item-specific vendors are listed below. Streptavidin-conjugatedhorseradish peroxidase (SA-HRP) was purchased from Thermo Scientific.When necessary, detection antibodies were biotinylated using a ThermoScientific EZ-Link NHS-PEG4-Biotin conjugation kit according to themanufacturer's protocol. A 4-chloro-1-naphthol (4-CN) solution waspurchased from Sigma-Aldrich. All other reagents were purchased fromThermoFisher, unless otherwise noted, and used as received.

Instrumentation: Sensor chips and instrumentation were obtained fromGenalyte (San Diego, Calif.), and their use has been describedpreviously (Washburn, A. L.; Gunn, L. C.; Bailey, R. C. Anal. Chem.2009, 81, 9499-9506). Briefly, UV photolithography and reactive ionetching were used to fabricate chip features on 8″ silicon wafers, priorto dicing into individual 6×6 mm chips. Each chip contains 32individually addressable microring resonators, 24 of which are used asactive biosensors and the other 8 serve as thermal controls. The sensorchip was sandwiched between an aluminum cartridge holder, 0.007″ Mylarflow gasket, and Teflon cartridge top to enable microliter-scale4-plexed fluidic delivery.

Biochemical Modification of Sensor Surface and Capture Agents: Prior tocovalent modification with S-4FB, antibodies were buffer exchanged into100 mM, pH 7.4 PBS using protein desalting columns, followed by a 2 hourincubation with a 10 fold molar excess of S-4FB. Unreacted S-4FB wasremoved using another desalting column.

Sensor chips were initially cleaned in a piranha solution (3:1 H₂SO₄/30%H₂O₂) for 30 seconds. Following a 3 minute sonication in ethanol, chipswere dried under N₂ and spotted with a 1 mg/mL HyNic silane solution for20 minutes, followed by another 3 minute sonication in ethanol anddrying with N₂. Chips were then loaded into the cartridge assembly and astable sensor response baseline in PBS 6 buffer was established.Antibody solutions (10 μg/mL) were flowed across the array at 10 μL/minfor 5 minutes before reducing the flow rate to 2 μL/min for another 25minutes. Following a 5 minute buffer rinse, chips were transferred to aStarting Block solution and stored overnight at 4° C. A representativesensor response during the antibody functionalization process is shownin FIG. 13.

Interleukin Assay: After antibody functionalization and overnightblocking, sensor chips were briefly rinsed in PBS 7.4 prior, loaded intothe cartridge assembly and then the instrument. All assay steps areperformed at a flow rate of 30 μL/min to minimize diffusion-limitedkinetics, and all sample/reagent injections are separated by 3 minutebuffer rinses. An initial 3 minute low-pH rinse was performed to removeany loosely bound blocking protein, followed by a return to a stablebaseline in PBS 7.4. Buffer solutions containing the interleukin sampleswere flowed across the array for 20 minutes. Subsequently, serialintroduction of biotinylated secondary antibodies (each 1 μg/mL) andSA-HRP (2 μg/mL) completes the surface bioconjugation. A freshlyprepared 4-CN solution (0.48 mM 4-CN; 0.01% H₂O₂; 17% methanol) was thenintroduced to the sensor for 25 minutes, and the HRP catalyzed oxidationof 4-CN resulted in the deposition of an insoluble precipitate of4-chloro-1-naphthon (4-CNP), which was detected as an increase in theresonant wavelength of the sensor. Preferably, the H₂O₂ was added to the4-CN solution immediately prior to its introduction to the sensor chip.It should be noted that sensor chips can be regenerated with minimaleffects on subsequent assay performance, however, only new sensor chipswere used in this study to eliminate such potential sources of error.

Cerebrospinal Fluid: Cerebrospinal fluid (Lot No. BCC062613PMG1) wasobtained from Chemed Services, and stored at −80° C. immediately uponreceiving. Samples were thawed at room temperature for 30 minutes priorto use, and introduced undiluted to the sensor surface at a flow rate of15 μL/min, to reduce sample consumption.

Data Analysis: All data analysis was performed using Origin Pro 9.0.Enzymatically-enhanced sensors responses were determined as thedifference in resonance wavelength shift before the addition of 4-CN andthen after a 5 minute buffer rinse following the 4-CN step. Controlrings functionalized with an IgG isotype control (anti-IL-4) weresubtracted from target-specific rings to correct for the bulk refractiveindex shift associated with the introduction of the 4-CN solution.Calibration data was fit to a logistic function:

$\begin{matrix}{{f(c)} = {\frac{A_{1} - A_{2}}{1 + \left( \frac{c}{c_{0}} \right)^{p}} + A_{2}}} & \lbrack 3\rbrack\end{matrix}$

where A₁ is the initial value limit, A₂ is the final value limit, c isthe center of the fit,

and p is the power of the fit.

Determination of Equilibrium Constant

Equilibrium dissociation constants were determined by fitting both theassociation and dissociation phases of interleukin binding to asurface-immobilized capture antibody with Equations S1 and S2,respectively:

f(t)=A−Ae ^(−k) ^(obs) ^((t−U))  [S1]

f(t)=A+Ae ^(−k) ^(obs) ^((t−U))  [S2]

where A is the y offset, k_(obs) is the observed binding constant, t isthe time, and U is the time offset. The dissociation rate constant,k_(d), is directly obtained from the dissociation phase ask_(d)=k_(obs). The association rate constant, k_(a), is obtained fromthe association phase by determining the slope of k_(obs) plottedagainst analyte concentration, as shown in Equation S3.

$\begin{matrix}{k_{obs} = {{k_{a}\lbrack A\rbrack} + k_{d}}} & \lbrack{S3}\rbrack \\{K_{ads} = \frac{k_{a}}{k_{d}}} & \lbrack{S4}\rbrack\end{matrix}$

TABLE 1 lists values for constants. TABLE 2 shows calibration fittingparameters. TABLE 3 lists sources of biologics.

TABLE 1 Constants Value k_(a) 2.8 × 10⁶M⁻¹s⁻¹ K_(d) 1.3 × 10⁻²s⁻¹  k_(ads) 2 × 10⁸M⁻¹  k_(d) 4.8 × 10⁻⁹M   

TABLE 2 IL-2 IL-6 IL-8 A₁ 11.27 −2.80 31.28 A₂ 11266.12 13125.6411685.34 C 218.10 621.47 226.15 P 1.36 1.08 1.28 Adj. R² 0.9866780.999946 0.995057

TABLE 3 Vendor Clone # IL-2 IL-4 IL-6 IL-8 Antigen 14-8029/ 34-8049/148069/ 208/IL/R&D eBioscience eBioscience eBioscience Systems Capture555051/BD 8-D4-8/ MQ₂-13A₅/ 554716/BD Antibody Biosciences eBioscienceeBioscience Biosciences Detection 555040/BD MQ₂-31C₃/ 554718/BD Antibody#1 Biosciences eBioscience Biosciences Detection MQ₁-17H₁₂/ BAF206/R&DBAF208/R&D Antibody #2 eBiosciences Systems Systems ELISA Kit BDBiosciences Abcam BD Biosciences

FIG. 1B shows the response from four representative microrings duringthe enzymatically enhanced detection of a 3125 pg/mL solution of IL-6.The primary, secondary, and tertiary binding responses for IL-6,secondary antibodies, and SA-HRP are all clearly visible in the insetbut are dwarfed by the large signal associated with the deposition of4-CNP, which leads to an ˜11000 pm shift in microring resonancewavelength. Notably, microrings functionalized with an IgG isotypecontrol (anti-IL-4) only show a bulk shift associated with theintroduction of the methanolic 4-CN solution, and they return to theirbaseline levels when the running buffer is introduced across the sensorarray, as indicated by the asterisk.

FIG. 17 is a scanning electron micrograph of a representative microringsensor used to acquire the data in FIG. 16 after the enzymaticenhancement step, clearly showing the presence of precipitated 4-CNP.SEM images from a microring used for a lower IL-6 concentrationdetection experiment, as well as a control microring, are shown in theSupporting Information. Higher-resolution SEM images reveal depositionsat distinct locations as small as 50 nm, which may be the result oflocalized precipitation from single enzymes; however, more investigationis needed to substantiate this hypothesis. Interestingly, the real-timemonitoring of 4-CNP deposition reveals a self-limiting enhancement after˜25 min, even in the presence of increasing concentrations of 4-CN. Wepropose that signal enhancement is ultimately limited by the physicalocclusion of the enzyme by the local accumulation of precipitate.

The advantages of enzymatic signal amplification on the microringresonator platform are readily apparent when compared to linear signalenhancement strategies previously employed. For comparison, a secondarydetection antibody alone enhanced the magnitude of the resonancewavelength shift relative to the primary antigen by ˜3, and a bead-basedapproach yielded an additional factor of ˜50 (Luchansky, M. S.; Bailey,R. C. Anal. Chem. 2010, 82, 1975-1981.; Kindt, J. T.; Bailey, R. C.Anal. Chem. 2012, 84, 8067-8074). In contrast, FIG. 16 illustrates aHRP-derived signal gain of 10⁴. Furthermore, this response is undernearly saturating concentrations; at lower antigen abundance, theamplification factor is even higher. However, at these lowerconcentrations the primary binding of the target antigen and evensecondary antibody are not even observable. The magnitude of thisresponse can be attributed to both the high turnover of HRP and thedense packing of the 4-CNP at the sensor surface, which overlapsstrongly with the evanescent optical mode of the resonator.

Quantitative Detection

To establish the quantitative detection capability of this signalenhancement strategy by analyzing solutions containing differentconcentrations of three different interleukin targets (IL-2, IL-6, andIL-8) were analyzed over a concentration range of 1 pg/mL up to 3125pg/mL. This represents a clinically relevant range, which includes bothbasal interleukin levels as well as elevated levels that are oftencorrelated with an active immune response. FIGS. 18A-18C shows therealtime monitoring of the 4-CNP deposition process following exposureof the microrings to IL-2, IL-6, and IL-8, respectively, across thisconcentration range. FIGS. 18D-18F show the calibration curves resultingfrom plotting the HRP-enhanced resonance wavelength shift as a functionof interleukin concentration. (Note that the calibration curves areplotted on a log-log plot for clarity.) For IL-2 and IL-6, the limit ofdetection, as defined by the response at 3σ of the baseline noisemeasured with control microrings, was 1 pg/mL, while the LOD for IL-8was 500 fg/mL. These assays also demonstrated a 3 order of magnitudedynamic range, which while already quite broad, could be furtherextended to higher concentrations via either sample dilution or byquantitation using the measured secondary or even primary responses, asdemonstrated previously (Luchansky, M. S.; Washburn, A. L.; McClellan,M. S.; Bailey, R. C. Lab Chip 2011, 11, 2042-2044). Combined, thisapproach would enable quantitation across 8 decades from 500 fg/mL to 50μg/mL without any alteration to the described assay protocol.

Detection in Biological Sample

After establishing important assay characteristics and metrics in acontrolled buffer system, the assay was applied to measurements in aclinically relevant sample matrix, cerebrospinal fluid (CSF). CSF is apromising biofluid for biomarker-based diagnostics of diseases anddisorders of the central nervous system as it is in direct contact withthe extracellular space in the brain and contains important putativediagnostic markers (Neurol. 2010, 6, 131-144; Olson, L.; Humpel, C. Exp.Gerontol. 2010, 45, 41-46; Fagan, A. M.; Holtzman, D. M. Biomarkers Med.2010, 4, 51-63; Hansson, O.; Zetterberg, H.; Buchhave, P.; Londos, E.;Blennow, K.; Minthon, L. Lancet Neurol 2006, 5, 228-234). Due to thepresence of the blood-brain barrier, CSF has been shown to have moredisease diagnostic value than plasma-based analysis for somebrain-related diseases (Llano, D. A.; Li, J. H.; Waring, J. F.; Ellis,T.; Devanarayan, V.; Witte, D. G.; Lenz, R. A. Alzheimer Dis. Assoc.Disord. 2012, 26, 322-328).

Aware that matrix effects could potentially interfere with quantitationin undiluted CSF, both external standard and standard additioncalibration methods were employed and the results were correlated withtraditional ELISA assays. It was anticipated that while standardaddition should better compensate for matrix effects to give moreaccurate measurements, the extrapolation necessitated in this methodmight lead to reduced precision (Ellison, S. L. R.; Thompson, M. Analyst2008, 133, 992-997). FIG. 19A shows good agreement between both theELISA and microring-based quantitation of IL-8, IL-6, and IL-2 in pooledhuman CSF samples when using external standard calibration. In allcases, the precision is comparable between the two technologies and themicroring measurements gave a somewhat lower concentration for IL-6 andIL-8. These discrepancies are likely due to complex matrix effects,which are known to differentially affect dissimilar detectiontechnologies (Khuseyinova, N.; Imhof, A.; Trischler, G.; Rothenbacher,D.; Hutchinson, W. L.; Pepys, M. B.; Koenig, W. Clin. Chem. 2003, 49,1691-1695). Importantly though, while variability between CSF samplesand processing methods prohibits a directly quantitative comparison, thequalitative order of abundance, IL-8>IL-6>IL-2, agrees with literaturereports (Llano, D. A.; Li, J. H.; Waring, J. F.; Ellis, T.; Devanarayan,V.; Witte, D. G.; Lenz, R. A. Alzheimer Dis. Assoc. Disord. 2012, 26,322-328.).

To further probe the accuracy of our enzymatically enhanced microringresonator measurements, a focused study on IL-6 was performed using themethod of standard additions to better compensate for matrix effects. Asshown in FIG. 19B, the implementation of the standard addition methodbrings the measured concentrations into excellent agreement. Notsurprisingly, the precision of the standard addition microringquantitation was reduced compared to the external standard calibration;however, an equivalent reduction in precision was noted for the ELISAmeasurement when subjected to an identical standard additionextrapolation. Data from standard additions are shown in FIG. 14.

Interleukins served as a challenging proof-of-principle system where lowlimits of detection are required for analysis within native biologicalsamples. However, the generality of this signal enhancement strategyshould lend it to be amenable to a wide variety of diagnostic targetsand assay panels that require multiplexed and relatively rapid analyses.For applications which require still further increases in sensitivity,this assay could be modified through the conjugation of a multitude ofenzymes to nanoparticles or other scaffolds or even the conjugation ofclusters of enzyme-functionalized nanoparticles.

An enzymatic signal enhancement strategy has been integrated with asilicon photonic sensing platform to enable the multiplexed detection ofinterleukins within undiluted cerebrospinal fluid. The HRP-catalyzedoxidation of 4-CN results in deposition of an insoluble product onto thesurface, which in turn elicits an incredibly large sensor response. Thehigh gain of this modular signal amplification strategy allowed for ≤1pg/mL limit of detection and a 3+ order of magnitude dynamic range in arelatively rapid (90 min) and multiplexed assay format. Comparison ofCSF interleukin levels measured simultaneously using this technologywere compared with individually performed ELISA assays and found to bein excellent agreement in terms of both accuracy and precision, helpingto further establish this technological platform for clinical diagnosticapplications.

The following references are each incorporated herein by reference intheir entireties.

REFERENCES

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The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

1. A method of detecting interleukin-6 (IL-6) comprising: (a) obtainingan optical ring resonator having a first anti-cytokine antibody attachedthereto, wherein the first anti-cytokine antibody is anti-IL-6 fromclone MAB206 antibody; (b) contacting the first anti-cytokine antibodywith a sample comprising IL-6 that selectively binds to theanti-cytokine antibody; (c) contacting the bound IL-6 with a secondanti-cytokine antibody that selectively binds to the IL-6, wherein thesecond anti-cytokine antibody is a biotinylated anti-IL-6 from cloneBAF206; (d) contacting the biolinylated second anti-cytokine antibodywith a streptavidin-horseradish peroxidase conjugate; (e) contacting thehorseradish peroxidase with a solution comprising 4-chloro-1-naphtholand hydrogen peroxide, under conditions that oxidize 4-chloro-1-naphtholto 4-chloro-1-naphthon, whereby the 4-chloro-1-naphthon precipitates onthe surface of the optical ring resonator; and (f) measuring a change inresonance wavelengths of the optical ring resonator, thereby indicatingthe presence of the IL-6.
 2. The method of claim 1, wherein a fluidiccell comprises the optical ring resonator.
 3. The method of claim 1,wherein step (e) comprises contacting the horseradish peroxidase with asubstantially continuous flow of the solution comprising the4-chloro-1-naphthol and hydrogen peroxide.
 4. The method of claim 3,wherein the 4-chloro-1-naphthon precipitates on the surface of theoptical ring resonator during the substantially continuous flow of thesolution.
 5. The method of claim 3, wherein the substantially continuousflow of the solution is maintained for steps (e) and (f).
 6. The methodof claim 3, wherein the flow rate of the solution is about 30 μL/min. 7.The method of claim 3, wherein a continuous flow of the solution ismaintained for step (e).
 8. The method of claim 7, wherein thecontinuous flow of the solution is maintained for steps (e) and (f). 9.The method of claim 7, wherein the flow rate of the solution is about 30μL/min.
 10. The method of claim 3, wherein a substantially continuousflow of reagents contacting the optical ring resonator having a firstanti-cytokine antibody attached thereto is maintained for each of steps(b), (c), (d), and (e).
 11. The method of claim 10, wherein the flowrate of the reagents is about 30 μL/min.
 12. The method of claim 3,wherein a continuous flow of reagents contacting the optical ringresonator having a first anti-cytokine antibody attached thereto ismaintained for each of steps (b), (c), (d), and (e).
 13. The method ofclaim 12, wherein the flow rate of the reagents is about 30 μL/min. 14.The method of claim 1, wherein the concentration of hydrogen peroxide isless than about 0.003%.
 15. The method of claim 1, wherein aconcentration of the IL-6 less than about 100 pg/ml is detected.
 16. Asystem for detecting interleukin-6 (IL-6) comprising: an optical ringresonator having a first anti-cytokine antibody attached thereto,wherein the first anti-cytokine antibody is anti-IL-6 from clone MAB206antibody; a sample comprising IL-6, wherein the IL-6 is selectivelybound to the anti-cytokine antibody; a second anti-cytokine antibodyselectively bound to the IL-6, wherein the second anti-cytokine antibodyis a biotinylated anti-IL-6 from clone BAF206; astreptavidin-horseradish peroxidase conjugate in contact with thebiolinylated second anti-cytokine antibody; a solution comprising4-chloro-1-naphthol and hydrogen peroxide in fluid communication withthe horseradish peroxidase, under conditions that oxidize4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the4-chloro-1-naphthon precipitates on the surface of the optical ringresonator; and a detector adapted to measure a change in resonancewavelengths of the optical ring resonator, thereby indicating thepresence of the IL-6.
 17. A system for detecting interleukin-2 (IL-2)comprising: an optical ring resonator having a first anti-cytokineantibody attached thereto, wherein the first anti-cytokine antibody isanti-IL-2 from clone MAB206 antibody; a sample comprising IL-6, whereinthe IL-6 is selectively bound to the anti-cytokine antibody; a secondanti-cytokine antibody selectively bound to the IL-6, wherein the secondanti-cytokine antibody is a biotinylated anti-IL-6 from clone 555051; astreptavidin-horseradish peroxidase conjugate in contact with thebiolinylated second anti-cytokine antibody; a solution comprising4-chloro-1-naphthol and hydrogen peroxide in fluid communication withthe horseradish peroxidase, under conditions that oxidize4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the4-chloro-1-naphthon precipitates on the surface of the optical ringresonator; and a detector adapted to measure a change in resonancewavelengths of the optical ring resonator, thereby indicating thepresence of the IL-2.